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					                           Biomolecular Chemistry 704



 Human Biochemistry:
NOTES & OBJECTIVES
                                    Fall 2005
                                     last updated 1/4/2007




                                      Christopher B. Kolar
                                       cbkolar@wisc.edu


This study guide has been created in the course of my studies at the University of Wisconsin
School of Medicine and Public Health. It is intended as an exam review of the required learning
objectives. It references a variety of course materials, including lecture, Power Point, assigned
readings, and sometimes outside sources. While I have attempted to make it as thorough, specific,
and accurate as possible, I cannot guarantee this, so use it at your own risk. If you have any
questions or comments, or have found an error within the text, please feel free to contact me.




                                                             COLOR KEY:
                                                             • red:            diseases
                                                             • blue:           medications
                                                             • orange:         enzymes and compounds
                                                             • pink:           microorganisms


                                                             FORMAT KEY:
                                                             • margins:   1”
                                                             • tab stops: 0.25”
                                                             • font:      Times New Roman
                                                             • size:      10
                                                                           Biochemistry: NOTES & OBJECTIVES (page 2 of 165)




1. Fundamentals of Protein Structure
TABLE – pK’ Values for Common R-groups
   group                                                approximate pK’
   α-carboxyl (FREE)                                    3 (C-terminal)
   β-carboxyl (Asp), γ-carboxyl (Glu)                   4
   imidazole (His)                                      6
   sulfhydryl (Cys)                                     8
   1˚ α-amino (FREE)                                    8 (N-terminal)
   2˚ α-amino (Pro, FREE)                               9 (N-terminal)
   ε-amino (Lys), phenolic hydroxyl (Tyr)               10
   guanido (Arg)                                        12
                                                                   [B  ]                  [HB ]
- Henderson-Hasselbalch equation:               pH  pK'  log            ; pH  pK'  log
                                                                   [HB]                     [B- ]
- modifications
   - phosphorylation:           attachment of a phosphoryl group to a hydroxyl group, extruding water
   - N-glycosylation:           attachment of a sugar to an amine (commonly with Asn)
   - O-glycosylation:           attachment of a sugar to an oxygen (commonly on Ser, Thr, modified residues)
   - hydroxylation:             attachment of a hydroxyl group to the R group (commonly on Pro, Lys)
   - carboxylation:             attachment of a carboxyl group to the R group (commonly on Glu)
- nomenclature
   - N-terminus to C-terminus
   - substitute –yl for –ine, except for aspartyl, asparaginyl, glutamyl, glutaminyl, cysteinyl, tryptophanyl
- peptide bond formation: carboxyl group + primary amine group  peptide bond + H2O
- protein structure
   - primary structure: sequence of amino acids
   - secondary structure: common organizations of structure
      - α-helix
         - 3.6 amino acids per helical turn, with each AA able to participate in up to two H-bonds
         - H-bonds: between =O, H-N- within helix, connecting i to i+4
         - little Pro due to incompatibility with helix angle; little Gly due to being too free to form tight conformations
      - β-pleated sheet
         - stretched polypeptide chains running either parallel or antiparallel, depending on chain orientation
         - H-bonds: between carbonyl and amide hydrogens of adjacent chains
      - β turn
         - allows protein backbone to make abrupt turns
         - abundant Pro, Gly, due to stearic considerations
   - tertiary structure: three-dimensional structure
   - quaternary structure: interaction of tertiary domains
- classes of proteins
   - enzymes:         accelerate the attainment of equilibrium
   - structural:      form biological structures
   - transport:       carry biochemically important substances
   - defense:         protect the body from foreign invaders




2. Enzyme Kinetics
- general features of catalysts
   - enzymes do not alter the final equilibrium ratio of substrates and products
   - enzymes act by lowering the activation energy of a reaction
                                                                          Biochemistry: NOTES & OBJECTIVES (page 3 of 165)



   - enzymes do not determine the direction of a reaction
   - catalysts are not used up during a reaction
   - enzymes have an active site that positions AA R-groups in the proper position for catalysis
- coenzymes and prosthetic groups
   - coenzyme: loosely bound (KD of 10-5 to 10-7)
   - prosthetic group: tightly bound (KD of 10-9, or covalently bound)
- enzyme activity
   - measurement of [product] vs. [time]
   - slope changes with the factor of the enzyme (for arbitrary units, m=2 with twice as much enzyme)




3. Factors Affecting Enzyme Activity
- constants
   - Vmax: maximum velocity of a reaction
     - extensive property (more enzyme  higher Vmax)
   - Km: Michaelis-Menten constant
     - intrinsic property (Km remains the same regardless of enzyme concentration)
     - substrate concentration at which enzyme is operating at half of V max
     - high Km: rate only approaches Vmax at high substrate concentrations
     - low Km: enzyme functions near Vmax even at lower substrate concentrations
- equations
                                                    Vm ax  [S]
  - Michaelis-Menten:                          v
                                                    K m  [S]
                                               1  K m  1     1
  - Lineweaver-Burke (reciprocal plot):           V  [S]   V
                                                     
                                               v  max        max
- inhibition
   - competitive inhibition: binds only to free enzyme (typically at active site)
      - Vmax:                 no effect
      - Km:                   raises apparent Km
      - saturation plot:      shifts Km to the right, graph approaches Vmax more slowly
      - reciprocal plot:      affects slope: rotates graph counterclockwise on the y-intercept
      - equation:             factor (1 + [I]/Ki) added to slope term
   - non-competitive inhibition: binds to E or ES complex (typically at distant binding site)
      - Vmax:                 decreases
      - Km:                   no effect
      - saturation plot:      flattens graph, but doesn’t affect rate of achieving Vmax
      - reciprocal plot:      affects slope and y-intercept: rotates graph counterclockwise on the x-intercept
      - equation:             factor (1 + [I]/Ki) added to slope AND intercept terms
   - uncompetitive inhibition: binds only to ES complex (typically at distant binding site); “self titrating”
      - Vmax:                 decreases
      - Km:                   decreases apparent Km
      - saturation plot:      flattens graph, makes it achieve Vmax much more quickly
      - reciprocal plot:      affects y-intercept: shifts graph leftward on a parallel line
      - equation:             factor (1 + [I]/Ki) added to intercept terms
   - irreversible inhibition: removes enzyme from the equation
      - Vmax:                 decreases stoichiometrically
      - Km:                   no effect
      - kinetics:             - at [I] = KI, looks similar to non-competitive inhibition
                              - however, at [I] = 2KI, gives 100% inhibition/deactivation, compared to 66.7%
                                                                        Biochemistry: NOTES & OBJECTIVES (page 4 of 165)




4. Cellular Strategies of Enzyme Regulation
- mechanisms of enzyme regulation
   - covalent modification
      - proteolysis: destroys or activates enzyme
      - R-group modification: phosphorylation, N-glycosylation, O-glycosylation, hydroxylation, etc.
   - feedback inhibition: final product inhibits first committed step of the pathway
   - allosteric regulation: changing enzyme shape through binding on an allosteric site
- allosteric regulation
   - features
      - allosteric enzymes almost always have more than one subunit
      - saturation kinetics is generally sigmoidal, rather than Michaelis-Menten (hyperbolic)
      - inhibitors push curve right (more extreme sigmoid); activators push curve left (more hyperbolic)
   - K0.5: threshold concentration for regulation
   - note that each individual enzyme is fully active or inactive
- aspartate transcarbamylase (ATCase): a model
   - reaction: aspartate + carbamoyl phosphate  N-carbamoylaspartate (ATCase, Pi)
   - function: introductory step in the production of CTP
   - structure
      - 12 subunits: 6 catalytic, 6 structural
      - arranged in two states: T-state (compressed, inactive) and R-state (open, active)
   - inhibitor: CTP; stabilizes T-state (pushes sigmoid curve right)
   - activator: ATP; stabilizes R-state (pushes curve left)
   - note that both ATP and CTP bind to the same site




5. Thermodynamics
- general considerations
   - thermodynamics indicates the favorability of a reaction given the conditions, NOT the speed or pathway
   - work can only be done if a system is not at equilibrium
- equilibrium constants
   - Keq=1: reaction is at equilibrium
   - Keq>1: products are favored
   - Keq<1: reactants are favored
- thermodynamics
                                                                                            [products]
  - free energy:           ΔG  ΔG' o  RT  ln(Q)         | G' o  -RT  ln(K eq ); Q 
                                                                                            [reactants ]
  - free energy (2):         ΔG  ΔH  T  S
  - gas constant (R): 1.987x10-3 kcal/mol K
  - enthalpy: a measure of the difference in heat between the products and substrates
  - entropy: a measure of the disorder between the products and the substrates
- ATP hydrolysis as an example
  - ATP +H2O  ADP + Pi
  - heavily skewed right
     - ATP is unstable due to the electrostatic repulsion
     - products are stabilized by ionization, resonance structures unavailable in ATP
     - products have greater entropy
  - considering the unique reactions in biochemist’s standard state, free energy is -4.5 kcal/mol
                                                                       Biochemistry: NOTES & OBJECTIVES (page 5 of 165)



- high energy bonds: those that have a free energy of -5 kcal/mol or lower
                                                                      Biochemistry: NOTES & OBJECTIVES (page 6 of 165)



- bond types
   - pyrophosphate:          PO3-2-OPO32-R                            high
   - phosphate ester:        PO3-2-OCH2                               low
   - carboxyl phosphate:     PO3-2-OOC                                high
   - enol phosphate:         PO3-2-OC(=CHR)-COOH                      high
   - guanido phosphate:      PO3-2-NH-C(=NH)-NH-R                     high
   - ester:                  R-O-C(=O)-R                              low
   - thioester:              R-S-C(=O)-R                              high
   - β-keto acid:            R-C(=O)-CH2-COO-                         high
   - glycoside/acetal:       R-O-CH-R-O (O connected to CH)           low
   - amide:                  NH2-C(=O)-R                              low
   - hemiacetal phosphate:   PO3-2-O-CH-O…                            ~high




6. Protein Folding and Dynamics
- the protein folding problem
   - protein primary structure contains all necessary information for the formation of tertiary structure
   - Levinthal paradox: proteins cannot possibly fold randomly, due to the time necessary
   - thus the protein folding problem is to figure out how the primary structure dictates the tertiary structure
- thermodynamics of protein folding
   - ΔGfold
      - plot % folding vs. [denaturant] to get two zero slopes with a measurable transition region
      - determine Keq within the transition region and plot ΔG vs. [denaturant] (Keq=(1-α)/α = [N]/[U]
      - extrapolate to the y-intercept to determine ΔGfold
   - implications of ΔGfold
      - most values for folding differ only by 5-10 kcal/mol
      - this indicates that proteins are dynamic structures, capable of moving and breathing as necessary in vivo
   - forces of binding
      - favoring native state
         - hydrophobic effect: removing water-fearing side chains from “solution” is energetically favorable
         - hydrogen bonding: secondary structure bonds are maximized, imparting stability
      - favoring unfolded state
         - conformational entropy: forcing one conformation is highly unfavorable
         - burying polar groups: some groups not involved in H-bonds are not buried, which is unfavorable
      - generally, ΔGfold is only slightly negative
   - structural implications
      - bond angles (Φ, Ψ) are only observed in very small Ramachandran space, which limits protein conformations
      - driving force of folding is the hydrophobic effect, so like groups cluster together
      - note that folded state of proteins is not solid, but rather dynamic
- folding in cells
   - chaperones: proteins that aid in protein folding
      - two major classes: Hsp70, chaperonins
      - either protect proteins from aggregation or actively assist in their folding
   - specific systems
      - protein disulfide isomerase: ensures that proper disulfide bonds are formed
      - peptide prolyl cis-trans isomerase: ensures that the most stable Pro configurations are made
                                                                         Biochemistry: NOTES & OBJECTIVES (page 7 of 165)



- protein misfolding
   - mutation: can cause reduced or non-function (e.g. CFTR transmembrane protein in cystic fibrosis)
   - multiple low-energy stable states: prions
     - consequences
        - often, only time is necessary for folding to the desired state
        - sometimes, protein misfolding can cause pathology, as in aggregation of prion plaques
        - in such cases, the misfolded protein can lead to formation of holes in brain tissue, leading to CNS problems
     - prion diseases
        - kuru
        - Creutzfeld-Jakob diseases
        - scrapie (sheep and goats)
        - mad cow disease (cows)
        - chronic wasting disease (deer)




7. Hemoglobin and Gas Transport
- oxygen transport
   - pO2: lungs, 100 torr; arteries, 95 torr; capillary bed, 20-40 torr
   - myoglobin
      - structure: single globin chain
      - function: O2 storage, release during very low O2 (θ=1-5 torr)
   - hemoglobin
      - structure: 2 α chains, 2 β chains, and noncovalently-bound heme (protoporyphrin IX, Fe(II))
      - function: oxygen transport (oxygen reduces Fe(II), which is reversible upon release of O 2)
      - regulation: cooperative (binding O2 increases chances of binding additional O2, same for release)
- factors affecting O2 transport
   - 2,3-bisphosphoglycerate (2,3-BPG): allosteric regulator
      - found in high concentrations in blood due to incomplete glucose metabolism
      - 2,3-BPG stabilizes T-state, pushing binding curve right and leading to O2 dump at higher pressures
   - Bohr effect: pH regulation
      - Hb + 4 O2  Hb(O2)4 + 2.4H+
      - thus if blood pH rises, O2 is dumped off more rapidly due to the Bohr effect (pushing the reverse direction)
      - pH rises: result from excess CO2, lactate production in working muscles
   - CO2 binding: leads to O2 dump
   - high temperature: leads to O2 dump
   - Gibbs-Donnan equilibrium effect: diffusible anions leave intracellular H + higher than extracellular
- CO2 transport
   - methods of transport
      - 10% as carbamino protein
      - 90% through bicarbonate-carbonic acid system
   - capillary bed: CO2 uptake, O2 dump
      - CO2 enters cell, is converted to carbonic acid, and then to bicarbonate plus hydronium
      - bicarbonate diffuses out of the cell and is replaced by Cl-
      - hydronium binds with free Hb to form HbH +, which draws HbO2 towards free Hb (O2 release)
      - O2 diffuses out of the cell
   - lungs: CO2 dump, O2 uptake
      - O2 enters cells, favoring the formation of HbO2, favoring the release of hydronium from HbH +
      - hydronium reacts with bicarbonate to form carbonic acid, which favors intake of bicarbonate and Cl - expulsion
      - bicarbonate is converted to CO2, which is expelled from the cell
- sickle cell anemia
   - caused by a G6V mutation in one of the Hb chains
   - this exposes a nonpolar surface patch, causing aggregation of Hb (and reduced functionality)
                                                                          Biochemistry: NOTES & OBJECTIVES (page 8 of 165)



  - aggregations impart sickle shape to cells, damaging them and affecting their ability to move in bloodstream
  - this disease leads to symptoms similar to anemia, is often fatal in childhood




8. Motor Proteins
- muscle contraction
   - structure
      - actin (thin filament): polar array of globular proteins arranged in a filament
      - myosin (thick filament): intertwined α-helix coiled coil with aligned head groups
   - muscle myofiber structure: Z I M H A
      - Z line: attachment point of actin thin filaments
      - M line: attachment point of myosin thick filaments
      - I: exclusively actin
      - H: exclusively myosin
      - M: intermixed actin and myosin
   - mechanism
      - release:               ATP binds, causing myosin to release from actin
      - extension:             ATP  ADP + Pi, causing the head group to extend slightly
      - binding:               Pi is released, causing the extended head group to bind
      - power stroke:          ADP is released, returning myosin to return to conformation (now moved slightly)
   - regulation
      - tropomyosin sits in the binding pocket, disallowing myosin binding to actin
      - upon CNS stimulation, Ca2+ stores in the cell are released
      - Ca2+ binds troponin C, which leads to a conformational change that moves tropomyosin
      - with sufficient ATP, muscle is able to begin power strokes
- P-loop NTPases
   - molecular motors using an NTP hydrolysis reaction to drive motor function typically use P-loops
   - P-loop: phosphate loop motif
      - binds the triphosphate moiety of the NTP
      - stabilizes the transition state for converting NTP to NDP + P i
      - frequently involves a lysine residue, metal ion, both of which stabilize the intermediate strong negative charge
- other molecular motors
   - kinesins: structural homologues of actin; transport cellular organelles on intracellular myosin-like microtubules
   - helicases: use NTP hydrolysis to unwind DNA




9. Defense and Structural Proteins
- antibodies
   - structure
      - two heavy chains linked by one or more disulfide bonds
      - each of two light chains linked by one disulfide bond
      - major classes: IgM, IgD, IgG, IgA, IgE (with IgG the most prominent at 75% of total)
   - function: binding to antigens to act as a signal for the immune response
   - binding strategies
      - globulin domains
         - heavy chain: VH, CH1, CH2, CH3
         - light chain: VL, CL
      - antibodies bind antigens at the N-terminal ends, at a variable site made of both light and heavy chain structures
         - hypervariable loop: variable domain antibody structures designed to bind antigens
                                                                      Biochemistry: NOTES & OBJECTIVES (page 9 of 165)



      - each antibody has three hypervariable loops that are almost perfectly matched to an antibody
      - note that antibodies can also be designed to bind small molecules using similar chemistry
- use in biochemistry
   - polyclonal antibodies: a heterogenous collection, each of which binds a different epitope (sequence) of antigen
   - monoclonal antibodies: homogenous collection that recognizes only one epitope of the antigen
                                                                           Biochemistry: NOTES & OBJECTIVES (page 10 of 165)



- collagen: an overview
   - collagen fiber: quarter-staggered array of cross-linked collagen triple helices
   - structural classification
      - group I:      type I, II, III, V         long polypeptide chains wound into a single helical domain
      - group II: type IV, VI, VII, VIII         long chains, several helical segments with several nonhelical segments
      - group III:                               shorter chains in one or more helical segments
   - synthesis
      - (1) synthesis of pro-α chain                                 ER/Golgi
      - (2) hydroxylation of selected Pro, Lys                       ER/Golgi
      - (3) glycosylation of selected hydroxylysine:                 ER/Golgi
      - (4) self assembly of pro-α chains                            ER/Golgi
      - (5) procollagen triple helix formation:                      secretory vesicle
      - (6) secretion:                                               plasmalemma
      - (7) cleavage of procollagen into tropocollagen:              extracellular matrix
      - (8) self assembly into collagen fibril:                      extracellular matrix
- collagen synthesis: detail
   - (1) pro-α chain structure: three polypeptide chains winding around a common axis
      - repeating sequence: Gly-X-Y, where X is commonly Pro, and Y is commonly HyPro
      - prevalence of Gly: allows R groups to fit at the center of the axis
      - prevalence of Pro, HyPro: imparts helical stability
   - (2) proline hydroxylation
      - prolyl residue + α-ketoglutarate  4-hydroxyprolyl residue + succinate
      - enzyme: prolyl 4-monooxygenase
      - monooxygenase enzymes
         - prolyl 3-monooxygenase, prolyl 4-monooxygenase, lysyl 5-monooxygenase
         - mechanism always require a second reducing agent (e.g. α-ketoglutarate)
      - cofactors: Fe2+, ascorbate (vitamin C)
         - vitamin C: reduces Fe2+ in hyxroxylase enzymes
         - lack of Vitamin C leads to scurvy, which is characterized by collagen weaknesses
   - (3) glycosylation: primarily galactose and glucosylgalactose; function unknown, may involve signaling
   - (4,5) procollagen
      - pro-α chains each have 100-300 extra amino acids at each end
      - with linkage by disulfide bonds, these stabilize the molecules during the formation of tropocollagen
   - (7) tropocollagen
      - formation
         - amino and carboxy procollagen peptidase enzymes cleave peptide bonds to release the precursor portions
         - this allows tropocollagen to self-assemble into insoluble collagen fobers
   - (8) maturation of the collagen fiber
      - during collagen maturation, lysyl oxidase catalyzes the formation of an aldehyde from lysine
      - critical step in forming cross-links; inhibition of this step can cause lathyrism, or defects in skeletal formation
- problems in collagen synthesis
   - lathyrism
      - inhibition of lysyl oxidase reaction in collagen maturation
      - characterized by defective skeletal formation, excretion of HyPro-rich peptides
   - Ehlers-Danlos syndromes
      - clinically heterogeneous category of connective tissue disorders
         - type IX: low lysyl oxidase activity; causes defective skeletal formation
         - type VII: decreased pro-collagen aminopeptidase activity, leading to fragile, loose, hyperextensible CT
         - type VI: deficiency in lysyl-5 monoxygenase, leading to fragile, loose, hyperextensible CT
      - types of Ehlers-Danlos syndromes listed here due to defects in modification, not synthesis, of collagen
   - breakdown
      - Clostridium histolyticum, which causes gas gangrene, contains a collagenase
      - mammalian tissues undergoing reorganization often have mammalian collagenases within
                                                                       Biochemistry: NOTES & OBJECTIVES (page 11 of 165)




10. Proteolysis in Blood Clotting
- digestion
   - zymogen: inactive form of the enzyme
   - trypsinogen  trypsin (enterokinase cleavage, activates trypsin only in intestinal lumen)
   - other pancreatic zymogens: chymotrypsinogen, procarboxypeptidase
   - serpin: serine protease inhibitor; antagonizes endopeptidases such as trypsin
- mechanisms of clot formation
   - vasoconstriction: release of serotonin by platelets
   - aggregation: mechanical obstruction of blood flow, stimulated by ADP released by platelets
   - cross-linked masses of protein: also restrict blood flow, and platelets again play a role
- hard clot formation
   - reactions
      - fibrinogen (α2β2γ2)  fibrin (α’2β’2γ2) [thrombin;  fibrinopeptides A, B]
      - fibrin (α’2β’2γ2)  fibrin soft clot (α’2β’2γ2)n [spontaneous aggregation]
      - factor XIII (a2b2 – plasma, a2 – platelet)  factor XIIIa (a’2) [thrombin, Ca2+, PF3;  activation peptides]
      - fibrin soft clot (α’2β’2γ2)n  HARD CLOT [XIIIa, a transglutaminase]
   - mechanism
      - fibrin formed by thrombin cleavage of fibrinogen to release fibrinopeptides A, B
      - fibrin spontaneously aggregates into a soft clot
      - factor XIII is activated by thrombin cleavage to release activation peptides
      - factor XIIIa, a transglutaminase, cross-links fibrin, initially at unreacted γ-chains, then α-chains
- thrombin formation
   - preprothrombin: synthesized in the liver
   - preprothrombin  prothrombin [reduced vitamin K, CO2]
   - prothrombin  thrombin [Xa, Va, Ca2+, PF3]
      - factor Va acceleration:         increases local Xa and prothrombin concentrations
                                        orients the enzyme and substrate molecules
                                        increases the Vmax of Xa
                                        alters conformation of prothrombin to make bonds more accessible
- clot formation diagram
   - intrinsic pathway: involving only factors found in the plasma
      - XI  XIa [XII]
      - IX  IXa [XIa, Ca2+, PF3]
      - X  Xa [IXa, Ca2+, PF3, VIIIa]
   - extrinsic pathway
      - VII + TF  VIIa · TF [Ca2+, PF3]
      - IX  IXa [VIIa·TF]
      - X  Xa [IXa OR VIIa·TF, Ca2+, PF3]
   - positive regulation
      - V  Va [thrombin]
      - VII  VIIa [Xa, Ca2+, PF3; OR thrombin]
      - VIII  VIIIa [Xa OR thrombin]
   - negative regulation
      - thrombin + fibrin  inactive thrombin/fibrin complex
      - VIIIa  VIIIi [ProteinCa, ProteinS; OR thrombin]
      - Xa  Xi [thrombin]
      - Va  Vi [ProteinCa, Protein S; OR thrombin]
      - prothrombin  inactive prothrombin [thrombin]
      - thrombin + thrombomodulin  thrombomodulin complex  activation of Protein C (Ca)
- prevention of clotting
   - protection of platelet integrity
   - removal of Ca2+ ions (useful in blood bank, not useful clinically)
   - heparin: serine protease inhibitor; antagonizes, IXa, Xa, XIa, XIIa
                                                                       Biochemistry: NOTES & OBJECTIVES (page 12 of 165)



   - vitamin K antimetabolites
   - protein C, protein S, thrombin-thrombomodulin system
- clot lysis
   - plasminogen  plasmin [tissue-type plasminogen activator (TPA)]
   - fibrin binds plasminogen, TPA; plasmin splits fibrin
- clot formation mechanisms
   - proteins activated by proteolytic cleavage:
      - VII, VIII, IX, X, XI, XII
      - prothrombin, fibrinogen, V, XIII, protein C
      - plasminogen
   - zymogens that become endopeptidases
      - VII, IX, X, XI, XIII
      - prothrombin, protein C
      - plasminogen
   - vitamin K-dependent factors: prothrombin, VII, IX, X, Protein C, Protein S
      - vitamin K used for carboxylation of glutamic acid residues
      - forms Ca2+-binding site that modulates binding to a phospholipid surface during activation
   - non-protein accelerator enzymes: Va, VIIIa, tissue factor
   - proteins deactivated by proteolytic cleavage: prothrombin, Va, VIIIa, Xa
- regulation of clot formation
   - localized, rapid response
      - enzyme cascade: per unit time, each enzyme can turn over many more products, increasing exponentially
      - localized binding, conc.: Vitamin K-dependent factors bind platelets via γ-carboxyglutamyl Ca2+ bridges
      - positive feedback: thrombin and Xa stimulate factors (V, VII, VIII) responsible for making thrombin and Xa
   - limiting the clot to the site of blood loss
      - platelet inversion causes release of serotonin (vasoconstrictor) and ADP (platelet aggregation)
      - factors continually present in blood and only activated locally, such as Vitamin K factors binding PF3
      - inactivation mechanisms: high [thrombin], thrombin/thrombomodulin, Protein Ca-Protein S complex
      - heparin: enhances antithrombin III (serine protease inhibitor), which antagonizes thrombin, IXa, Xa, XIa, XIIa
      - thrombin binds fibrin to form an inactive complex
   - thrombin-based clot slowing
      - cleaves factors Va, VIIIa, Xa, prothrombin
      - interacts with thrombomodulin to activate protein C, which complexes with S to inactivate Va, VIIIa
      - forms an inactive complex with fibrin
      - heparin: enhances antithrombin III (heparin), which antagonizes factors thrombin, IXa, Xa, XIa, XIIa




11. Drug Interactions with Enzymes
- HIV protease
   - HIV protease is an aspartyl protease critical to the cleavage of polyproteins
   - Crixivan: binds to the active site as a classic competitive inhibitor
- COX inhibitors
   - prostoglandin H2 synthase-1 is involved in formation of prostaglandins, which are part of regulation of pain
   - aspirin: suicide inhibitor (covalent acetylation of a key serine active site residue)
   - ibuprofen: competitive inhibitor (transition state mimic)
- penicillin: suicide inhibitor
   - blocks the formation of glycopeptide transpeptidase in bacterial cell wall formation
   - acts as a substrate mimic to form an acyl intermediate, but with an irreversible bond
                                                                          Biochemistry: NOTES & OBJECTIVES (page 13 of 165)




12. Proteomics and Methods in Protein Science
- separation techniques
   - protein precipitation: salting out
      - addition of salts [e.g. (NH4)2SO4] to precipitate a protein
      - different proteins are sensitive to different concentrations
   - separation by surface charge: ionic exchange chromatography
      - solution poured through a column of ion exchange beads, and proteins differentially associate
      - ionic attractions are reversed by addition of salt solutions, which proteins are also sensitive to
   - separation by size: size exclusion chromatography
      - proteins are poured through a column of porous beads
      - larger proteins skip the pores and come off first; smaller proteins remain behind longer
   - separation by affinity: affinity-based chromatography
      - chromatographic beads can be made with specific ligands in place
      - those proteins binding the ligand will remain behind, and bona fide ligand can be used to wash from column
- analysis and identification techniques
   - gel electrophoresis
      - proteins are denatured and given a high negative charge by sodium dodecyl sulfate (SDS)
      - proteins are loaded onto an electrophoretic gel, dyed, and an electric charge is applied
      - larger proteins will not migrate as far as smaller proteins
   - Western blotting
      - visualized antibodies used to specifically detect a certain protein off of an SDS gel
      - this allows the determination of protein location in even a very complex protein mixture
   - mass spectroscopy: determination of mass of a protein or its peptides
- proteomics
   - proteomics: the study of the entire protein complement of an organism
   - tandem affinity purification
      - protein is artificially tagged by two proteins that have a high affinity for purification beads
      - tagged protein is expressed in cells
      - protein mixture is eluted through column that binds tag #2 beads
      - TEV protease used to remove complexes from column, and mixture eluted through tag #1 column
      - protein identification proceeds by mass spectrometry




13. Inheritance of Genes
Study Guide
do the following:
- mitotic and meiotic divisions
   - mitotic
      - DNA replication: forms two copies (sister chromatids) of each homolog                          46 x 2
      - division:             sister chromatids are separated, making identical diploid cells          46 x 1
   - meiotic
      - DNA replication: forms two copies (sister chromatids) of each homolog                          46 x 2
      - division 1 (meiotic): homologous chromosomes are separated                                     2 x (23 x 2)
      - division 2 (mitotic): sister chromatids are separated, making varied haploid cells             4 x (23 x 1)
- definitions
   - homozygous: diploid individual has identical alleles at a given locus
   - heterozygous: diploid individual has different alleles at a given locus
   - genotype: genetic composition of an individual
   - phenotype: observable characteristics of an individual resulting from the genotype
   - dominant: trait seen in individuals heterozygous for the causative allele
   - recessive: trait seen only in individuals homozygous for the causative allele
                                                                         Biochemistry: NOTES & OBJECTIVES (page 14 of 165)



   - autosomal: gene on one of the 22 autosomes
   - X-linked: gene on the X chromosome
- recognizing patterns of inheritance
   - autosomal dominant: found in each generation, afflicting on average half of progeny
   - autosomal recessive: seemingly skips generations, appearing on average in 1/4 of progeny of unafflicted
   - sex-linked: appears commonly in sons of unafflicted; females require an afflicted father and carrier mother
- genetic linkage
   - Mendelian inheritance requires two individual traits to separate independently (50% separation)
   - traits that are physically very close on a chromosome will often separate with one another
   - the distance between traits is thus measured by separation using cM, where 1 cM = 1% separation
you should know:
- crossing over (meiotic recombination)
   - occurs during division 1 of meiosis, when homologous chromosomes line up on one another
   - occurs 1-2 times per homologous chromosome pair
- reasons for patterns of inheritance
   - autosomal dominant: manifestation of disease requires only one copy of afflicted trait
   - autosomal recessive: manifestation of disease requires two copies of the defective trait
   - sex-linked: must be homozygous for mutation, but males have only one copy of the chromosome
you should understand:
- X-inactivation
   - expression of genetic material requires a careful balance, one that is disrupted in the sex chromosomes
   - to compensate, cells in early females inactivate one of the X chromosomes
   - reactivation occurs only when gametes are being formed
- linkage and recombination
   - recombination events can occur anywhere on a chromosome, so any two traits could separate independently
   - traits that are very close together have a statistically smaller chance of being split by a recombination event
   - traits at opposite ends of a chromosome are essentially independent


Notes: Lecture and Reading
characteristics of the human genome
- overview of the human genome
   - genome: complement of genetic information stored in an organism’s DNA
   - length (haploid number): 3 x 109 base pairs
   - number of genes: 35,000 (twice as many as in the round worm)
- humans as diploid organisms
   - homologs: maternal and paternal copies of a chromosome containing similar – but not identical – sequences
   - alleles: exact form of a gene at a given locus
   - thus each human cell has two homologs of the 22 autosomes and two sex chromosomes, for a total of 46
- chromosomes
   - locus: position of a gene in the genome
   - chromosome: segments of a genome
   - human genome: 24 chromosomes; 22 are autosomal, the other two are X and Y
   - chromosomes average 1000 – 2000 loci each
   - sister chromatids: identical copies of a chromosome that exist after replication in preparation for division
   - centromere: attachment point of sister chromatids post replication
- mitochondrial chromosomes
   - mitochondria contain multiple copies of a chromosome that code for ~35 mitochondrial proteins or RNA
   - maternally inherited: sperm cells are devoid of cytoplasm
   - certain myopathies and neuropathies result from mutations in the mitochondrial chromosome
gametogenesis, recombination, and linkage
- germ line
   - germ line: the collection of cells that give rise to gametes (eggs and sperm)
   - somatic cells: all other cells of the body
   - only mutations in germ line cell DNA are passed onto offspring
- division
                                                                    Biochemistry: NOTES & OBJECTIVES (page 15 of 165)



   - mitosis: (46 x 2)  (46 x 1), (46 x 1)
      - division: sister chromatids separate
   - meiosis: (46 x 2)  (23 x 2), (23 x 2)  (23 x 1), (23 x 1), (23 x 1), (23 x 1)
      - division 1: homologous chromosomes separate
      - division 2: sister chromatids separate (similar to meiotic division)
- recombination
   - crossing over: meiotic recombination
   - occurs once or twice per chromosome pair, resulting in patchwork chromosomes from maternal, paternal DNA
- linkage
   - linkage: the tendancy of an allele at one locus to end up in the same gamete as another locus
      - tightly linked: combination of alleles corresponds to combination in either of the parental homologs
      - unlinked: equal proportions of all possible combinations are observed
   - loci on different chromosomes are unlinked
   - loci on the same chromosome are linked in proportion to their physical separation
      - 1 cM: 1% chance of being separated by recombination (works to ~50 cM due to probability)
      - average human chromosome is 150 cM long, so opposite ends are unlinked
general aspects of inheritance
- definitions
   - homozygous: diploid individual has identical alleles at a given locus
   - heterozygous: diploid individual has different alleles at a given locus
   - genotype: genetic composition of an individual
   - phenotype: observable characteristics of an individual resulting from the genotype
   - dominant: trait seen in individuals heterozygous for the causative allele
   - recessive: trait seen only in individuals homozygous for the causative allele
   - autosomal: gene on one of the 22 autosomes
   - X-linked: gene on the X chromosome
- limitations
   - this course considers only single gene defects exhibiting simple Mendelian inheritance
   - over 3000 have been identified
      - half are autosomal dominant
      - one third are autosomal recessive
      - less than one tenth are X-linked
autosomal inheritance
- autosomal dominant
   - transmitted only to offspring that have at least one afflicted parent
   - both males and females suffer, and about half of the individuals from a parent are affected
- autosomal recessive
   - can occur in the offspring of unafflicted parents if both are carriers (P=0.25)
   - males and females afflicted with equal probability
X-linked inheritance
- inheritance
   - disease is fully expressed in males due to being hemizygous
   - normal male, carrier female
      - son: 50% chance of being afflicted
      - daughter: 50% chance of being a carrier
   - afflicted male, normal female
      - daughter: always at least a carrier if mother is normal
      - son: always normal
- X inactivation
   - to maintain genetic balance, females have one X chromosome inactivated during early fetal development
      - this is random within a particular cell
      - however, this relationship is maintained within subsequent mitotic divisions
   - each cell can express only one X allele of an X-linked locus, though on average they are equally expressed
                                                                    Biochemistry: NOTES & OBJECTIVES (page 16 of 165)




14. Structure of Genes
Study Guide
do the following:
- ribose and deoxyribose
   - ribose:         5-carbon sugar, with 1’C ester-linked to 4’C
                     5’C: methyl group facing up off the sugar backbone
                     OH groups: 1’ (up), 2’ (down), 3’ (down), 5’
   - deoxyribose: ribose with –OH group removed from 2’C
- nucleotide attachments
   - base:           replaces –OH group on 1’C
   - phosphates: phosphate ester linkage to 5’C
- base recognition
   - purines: double ring structures
      - adenine: amino group
      - guanine: carboxy group
   - pyrimidines: single ring structures (like a hexagonal “pyramid”)
      - cytosine: amino group, carboxy group, methyl group
      - thymine: 2 carboxy groups, methyl group
      - uracil: 2 carboxy groups
- abbreviations (N = nucleotide base, d=deoxy)
   - nucleoside: N; dN
   - nucleotide: NTP (3 phosphates), NDP (2 phosphates), NMP (1 phosphate); dNTP, dNDP, dNMP
- polynucleotide polarity
   - 5’ end: phosphate molecule attached to 5’ carbon of sugar
   - 3’ end: -OH attached to 3’ carbon of sugar
   - by convention, DNA is written 5’ to 3’
you should understand:
- stabilization of complementary bases in double-stranded DNA
   - hydrogen bonds: A=T and G≡C complementary pairing
   - hydrophobic interactions: base stacking between bases of adjacent nucleotides
- orientation of DNA strands: antiparallel (5’  3’ runs with a 3’  5’ strand)
- separation of the strands
   - denaturation: heat (~100 °C, chemical denaturants, or DNA helicases can split the DNA strands
   - renaturation: strands come together as complementary base pairing regions find one another
      - reannealing: strands were previously paired
      - hybridization: strands were not previously paired
- use of a probe
   - probes are short oligonucleotide sequences that complement small regions of DNA
   - if mixed in excess with denatured DNA, they will find complements faster than the larger complete strands
   - this can be used in numerous genetic tests, as well as PCR
be able to:
- enzymatic reaction of DNA polymerase
   - deoxynucleotide triphosphate to be added is brought onto the template strand on the 3’ end of the primer
   - 3’ –OH attacks the 5’ dNTP at the α phosphate, extruding pyrophosphate and forming the phosphodiester bond
   - pyrophosphate is broken down into 2 phosphates by phosphatase, which helps to drive the reaction


Notes: Lecture and Reading
the central dogma of gene expression
- genotype  phenotype (through gene expression)
   - central dogma: DNA  RNA (transcription)  protein (translation)
   - some RNA does not make protein
- components
                                                                      Biochemistry: NOTES & OBJECTIVES (page 17 of 165)



   - RNA polymerase: transcribes DNA to make RNA
   - mRNA (messenger): class of RNA that is used to make protein
   - ribosome: translates RNA into protein
   - tRNA (transfer): class of RNA that supplies appropriate amino acids for addition to the growing chain
- additional complexities in eukaryotes
   - transcription: occurs in the nucleus
   - translation: occurs in the cytoplasm
   - introns: extraneous internal segments of RNA that are removed prior to translation by splicing
- gene expression
   - direct: level of transcription
   - indirect: post-transcriptional
- DNA replication
   - enzyme: DNA polymerase
   - each strand is copied, producing two identical double-stranded DNA molecules
DNA and RNA as nucleotide polymers
- nucleotide structure
   - 5-carbon sugar:          ribose in RNA, deoxyribose in DNA
   - base:                    purine or pyrimidine; attached to 1’ carbon of the sugar
   - phosphate:               one or more; attached to the 5’ carbon of the sugar
- nucleoside structure: nucleotide without phosphate
- nucleotides in RNA
               sugar              base              type              nucleoside        nucleoside     nucleotide
                                                                                       abbreviation   abbreviation
          ribose             adenine         purine               adenosine           A             AMP
          ribose             cytosine        pyrimidine           cytidine            C             CMP
          ribose             guanine         purine               guanosine           G             GMP
          ribose             uracil          pyrimidine           uridine             U             UMP

- nucleotides in DNA
             sugar            base              type             nucleoside        nucleoside         nucleotide
                                                                                  abbreviation       abbreviation
        deoxyribose       adenine        purine               deoxyadenosine      dA               dAMP
        deoxyribose       cytosine       pyrimidine           deoxycytidine       dC               dCMP
        deoxyribose       guanine        purine               deoxyguanosine      dG               dGMP
        deoxyribose       uracil         pyrimidine           deoxythymidine      dT               dTMP

- nucleic acid structure
   - phosphodiester linkage: joined by covalent bond between 3’ carbon of one, 5’ carbon of next
   - polarity: by convention, DNA is written from 5’ to 3’, reflecting the direction of synthesis of DNA and RNA
      - 5’ end: usually has a phosphate attached
      - 3’ end: usually has a hydroxyl attached
DNA as a double stranded helix
- general structure
   - external phosphate backbone, internal nucleotide bases
   - predominantly a right-handed antiparallel double helix
- stabilizing interactions
   - hydrophobic interactions between aromatic ring structures of bases
   - hydrogen bonds between donor and acceptor groups on the bases
      - specific interaction: only A=T and G≡C bonds are stable due to complementary nature of bases
      - genetic information is thus stored as a base pair sequence and is inherently redundant
separation of the two strands
- denaturation of noncovalent interactions
   - heat:                     e.g. boiling in aqueous solution)
   - chemical denaturants: e.g. urea, which competes for H-bonds)
   - DNA helicase:             ATP-based enzyme that can unwind DNA at ambient temperatures
                                                                       Biochemistry: NOTES & OBJECTIVES (page 18 of 165)



- melting temperature Tm
   - Tm: temperature at which DNA will denature into two single strands
   - higher proportion of G≡C: higher Tm, as there is a greater number of H-bonds
   - salt solutions: higher Tm, as salt shields repulsive forces between phosphate groups on opposite strands
- renaturation
   - reannealing: strands were previously paired
   - hybridization: strands were not previously paired
      - a short fragment added in excess will find complementary regions faster than the original strand
      - this is the basis for PCR, many genetic tests
DNA replication
- cell cycle
   - M phase:         mitosis and creation of a daughter cell
   - G1 phase:        gap phase and preparation for replication
   - S phase:         DNA replication
   - G2 phase:        gap phase and preparation for cell division
- replication
   - helicase unwinds DNA into its leading and lagging strands
   - leading strand synthesis: continuous line in 5’  3’ direction
   - lagging strand synthesis
      - primase adds an RNA primer
      - polymerase III synthesizes an Okazaki fragment
      - polymerase I replaces RNA with DNA
      - ligase closes Okazaki fragments, giving the complete strand form
- bond formation
   - DNAp promotes base pairing of appropriate dNTP to template DNA
   - DNAp catalyzes attach of 3’-OH on phosphate of the dNTP, forming a phosphodiester bond and releasing PP i
   - pyrophosphatase hydrolyzes PPi, helping drive DNA synthesis
- proofreading
   - DNA polymerase has a 3’5’ exonuclease activity, which adds to DNA fidelity
   - fidelity still results in a mistake in 1 per 108 nucleotides
- SUMMARY: enzymes of DNA replication
   - DNA helicase: unwinds DNA at the replication fork (ATP-driven)
   - DNA polymerase (DNAp)
      - phosphodiester bond formation: catalyzes addition of nucleotides to 3’-hydroxyl end of primer (5’3’)
      - 3’-5’ exonuclease activity: stops at mismatched bases and releases a dNMP
      - RNA replacement: replaces RNA fragments with DNA
   - RNA primase: adds RNA primers for use by DNAp
   - pyrophosphatase: hydrolyzes pyrophosphate (PPi + H2O  2Pi), helping drive bond formation by mass action
   - DNA ligase: forms phosphodiester linkage between Okazaki fragments




15. Expression of Protein-Coding Genes
Study Guide
be able to:
- RNA polymerase
  - non-template (RNA-like) strand: split off of DNA at replication fork, and is not read
  - template strand: proceeds through at active site, and is read in a 3’  5’ direction
  - growing mRNA: synthesized in a 5’  3’ direction, as in DNA replication
  - NTP substrate: added to 3’ end of growing mRNA chain, as in DNA replication
  - enzymatic reaction: chemically similar to DNA replication
- RNAp vs. DNAp
  - similarities: similar chemical reactions, direction of synthesis
                                                                      Biochemistry: NOTES & OBJECTIVES (page 19 of 165)



   - differences: RNAp does not require a primer; DNAp synthesizes two new strands simultaneously
- eukaryotic mRNA
   - cap: 7-methylguanosine triphosphate (m7Gppp) linked in a 5’-5’ linkage at the upstream end of the mRNA
   - 5’-UTR: upstream region prior to start site; plays roles in protein synthesis and mRNA degradation regulation
   - start codon: AUG; in eukaryotes, typically the most upstream AUG codon in the sequence
   - protein coding region: contains region coding protein (in mature mRNA)
   - 3’ UTR: region after a stop codon; plays roles in mRNA degradation
   - poly(A) signal: AAUAAA at end of 3’ UTR
   - poly(A) site: point in transcript where cleavage and polyadenylation occurs
   - poly(A) tail: AAAAAAn region that forms the most downstream end of the mRNA
- consequences of mutations in β-globin gene (β-thalassemia)
   - promoter regions: point mutations generally result in β+ alleles
   - 5’ splice site of intron 1:        mutation in initial GU sequence results in β0 allele
                                        mutation in more weakly-conserved downstream site results in β+ allele
   - 3’ splice site of intron 2:        mutation in terminal AG results in β0 allele
   - poly(A) signal: mutations cause a loss of mRNA stability, resulting in a β+ allele
   - nonsense mutations: β0 allele
   - frameshift mutations: β0 allele
   - missense mutations: rarely affect levels of β-globin produced, so do not result in thalassemia
you should know:
- RNA functions:
   - mRNA: template for protein synthesis
   - tRNA: carry amino acids to nascent polypeptides
   - snRNA: involved in pre-mRNA processing
- composition and function of the ribosome
   - large subunit (60S) and small subunit (40S)
      - 40S subunit: decoding of mRNA
      - 60S subunit: carries out peptidyl transferase reaction
   - three sites
      - A site: aminoacyl tRNA site, containing the incoming peptide
      - P site: peptidyl tRNA site, containing the growing polypeptide chain
      - E site: exit site, where empty tRNA molecules leave the ribosome
   - function: to read an mRNA and produce a protein
- composition and function of the spliceosome
   - composition: U1 snRNA (and four others) and associated snRNPs
   - function: splicing out of introns from pre-mRNA
- codons and anticodons
   - codon: read off of template strand of mRNA in 5’3’ direction
   - anticodon: complements codon and allows charged tRNA to insert correct amino acid in nascent polypeptide
- mutations
   - nonsense mutation: inserts a stop codon, resulting in a truncated protein
   - missense mutation: changes a codon so that it specifies a different amino acid
   - frameshift mutation: changes reading frame of mRNA, often resulting in downstream stop codons


Notes: Lecture and Reading
basic mechanism
- RNA polymerase (RNAp)
  - similarity to DNAp: single-stranded DNA template directs 5’  3’ synthesis of a polynucleotide chain
  - difference: does not require a primer
- mechanism of RNAp
  - initiation
     - promoter: short DNA sequences recognized by RNAp and its accessory proteins
        - orientation of promoter determines orientation of RNAp
        - this determines which strand is the template strand
     - RNAp binds at the promoter, and a small region of double-stranded DNA is denatured
                                                                      Biochemistry: NOTES & OBJECTIVES (page 20 of 165)



      - base-pairing guides polymerization except that an A in template DNA specifies incorporation of U in RNA
   - elongation
      - growing polynucleotide chain continually displaced from template, so DNA/RNA duplex is short
      - as RNA is displaced, DNA duplex is reformed
   - termination
      - RNAp or a termination factor recognizes a specific sequence or secondary structure in the transcribed RNA
- transcription units
   - transcription unit: a region of DNA containing a promoter and a termination signal, thus directing synthesis
      - in eukaryotes, this is typically equivalent to a gene
      - in bacteria, several genes often contained within a single transcription unit (operon)
   - transcript: the RNA made from a transcription unit
      - transcript sequence analogous to sequence of the non-template strand
      - difference: replacement of T with U
messenger RNA and the genetic code
- overview
   - codon: group of three adjacent nucleotides specifying the amino acid of a protein
   - untranslated regions (UTRs): portions at 5’ and 3’ ends that are not translated
      - range in length from <50 to several thousand nucleotides
      - play a role in regulation of translation, degradation of mRNA
   - primary structure of mRNA
      - methylguanosine cap
      - 5’ UTR
      - translated region
         - AUG (start) codon
         - protein-coding region (may contain introns that are excised prior to translation)
         - stop codon
      - 3’ UTR
         - poly-A signal (AAUAAA)
         - poly-A site
         - poly-A tail
- post-transcriptional modifications
   - methylguanosine cap (m7G)
      - 5’ triphosphate remains on the first codon
      - shortly after leaving RNAp II, capping enzymes add a methylated guanosine in a 5’-5’ triphosphate linkage
         - this protects from degradation by 5’ exonucleases
         - this also is a binding site for proteins that facilitate:
            - splicing of the pre-mRNA
            - transport of the mRNA to the cytoplasm
            - translation of the mRNA by ribosomes
   - termination and the poly-A tail
      - nascent polynucleotide is cleaved 10-30 nucleotides downstream of 5’-AAUAAA-3’
      - poly(A) polymerase then adds 100-200 AMP residues to the 3’ end, using ATP and releasing PP i
         - this poly(A) tail is found only on mRNA
         - thought to inhibit 3’-exonucleolytic degradation, promote translation
      - poly(A) site: point in the transcript where cleavage and polyadenylation occurs
      - poly(A) signal: AAUAAA sequence
   - mRNA splicing
      - definitions
         - pre-mRNA: precursor to eukaryotic mRNA, prior to excision of intervening sequences
         - intron: intervening sequences that interrupt mRNA-coding sequences
         - exon: mRNA-coding sequences, not necessarily protein-coding
         - RNA splicing: the process of intron removal; occurs in the nucleus in the spliceosome
      - process
         - spliceosome composed of pre-mRNA substrate, 5 snRNAs, and >60 proteins
         - spliceosome base pairs with sequences in the intron to direct splicing
         - released intron is degraded and nucleotides are recycled, while mRNA is transported to the cytoplasm
                                                                       Biochemistry: NOTES & OBJECTIVES (page 21 of 165)



      - introns in a gene
         - can vary from 0 to >50, though most genes have several
         - example: Factor VIII gene is 186 kbp in length, and 175 kbp is contained in 25 introns
         - first exon always contains at least some of the 5’-UTR
         - last exon always contains at least some of the 3’-UTR, including the poly(A) signal
- the genetic code
   - overview
      - 64 trinucleotide codons to specify each of the 20 amino acids
      - one start codon (AUG, or methionine), and three stop codons (UAA, UAG, UGA)
      - when an amino acid is specified by multiple codons, the 3 rd codon is often the only difference
   - mutations
      - silent mutation: does not change the sequence of the encoded protein
      - missense mutation: changes the codon to specify a different amino acid
      - nonsense mutation: changes a codon to a stop codon
      - frameshift mutation: deletion of addition of nucleotides such that the downstream reading frame shifts
point mutation in β-thalassemias
- thalassemias: an overview
   - thalassemia: inherited deficiency in the production of α or β globin (resulting in α or β thalassemia)
   - the globin chain present in normal amounts tends to form insoluble homotetramers that do not function well
   - when either chain is nearly absent, severe anemia and death usually occur before 10 without regular transfusions
- variance in severity
   - β0: alleles that are completely inactive
   - β+: alleles that are partially active
   - thalassemia minor: β0β, asymptomatic
   - thalassemia major: β0β0, requires regular blood transfusions
   - intermediate: β+β+, shows intermediate symptoms
- mutations and consequences
   - promoter regions
      - cluster in two regions, about 90 and 30 base pairs upstream of transcription start site
      - CACCC at -88: regulatory protein
      - ATA at -31: TATA box for binding TFIID
      - point mutations generally result in β+ alleles, with transcription reduced ~5-fold
   - splice sites
      - 5’ splice site of intron 1
         - mutation in initial GU sequence results in β0 allele
         - mutation in more weakly-conserved site at position 5 results in β+ allele
      - 3’ splice site of intron 2
         - mutation in terminal AG to GG mutation results in β0 allele
   - poly(A) signal: mutation of AAUAAA to AACAAA
      - results in cleavage of pre-mRNA after another AAUAAA sequence 900 nt further downstream
      - less β-globin is made due to a loss in stability, resulting in a β+ allele
   - other mutations
      - nonsense mutation in exon 2: β0 allele
      - frameshift mutations: β0 allele
      - missense mutations: rarely affect levels of β-globin produced, so do not result in thalassemia
         - exception: Indianapolis β-globin, which is highly unstable due to a single AA substitution
         - generally, may result in pathological effects, such as sickle cell anemia
stable RNAs with biochemical functions
- translated vs. untranslated RNA
   - mRNA makes up a small fraction of a cell’s RNA (10%)
   - most RNA in a cell is not translated, instead serving important cellular functions
- ribosomal RNA (rRNA) (75% of total cellular RNA)
   - the ribosome is 2/3 RNA and 1/3 protein
   - catalysis of peptide bond formation by modern ribosomes is carried out by rRNA
- transfer RNA (tRNA) (15% of total cellular RNA by weight)
   - used by ribosome to read mRNA codon, provide the corresponding amino acid
                                                                         Biochemistry: NOTES & OBJECTIVES (page 22 of 165)



      - one end: anticodon complementary to a given codon
      - other end: amino acid corresponding to a codon
   - undergo post-translational modification, giving them bases other than A, C, G, U (e.g. T by methylation)
   - due to redundancy, more than 20 different kinds of tRNAs
- small nuclear RNA (snRNA)
   - small, 100-300 nucleotide RNAs that participate in processing of pre-mRNA in the nucleus
   - packaged with proteins into small nuclear ribonucleoprotein particles (snRNPs)
   - some autoimmune disorders (e.g. systemic lupus erythematosis) produce antibodies that recognize snRNPs
      - the disease significance of this is not known
- small nucleolar RNA (snoRNA)
   - similar to snRNA, but found in nucleolus, which is dedicated to ribosome synthesis
   - base pair with newly-synthesized rRNA, direct processing and modification of rRNA into mature form
   - rRNA then assembles with ribosomal proteins to form the ribosomal subunits, sent to cytoplasm
- micro RNA (miRNA)
   - 20-22 nucleotides long, complementary to specific mRNA
   - pairing of miRNA with mRNA targets mRNA for degradation by ribonucleases
   - have an important role in human gene expression




16. Genetic Screening
Study Guide
- detection of sickle cell anemia
   - carrier detection: place blood droplets in low O2 environment, microscopically look for sickle cells
   - prenatal detection
      - recognize that sickle cell mutation destroys an MstII restriction enzyme site
      - digest the allele DNA with MstII and place it in a size-based gel electrophoresis
         - normal individuals will have a long (1.15 kb) and a short (0.2 kb) fragment
         - afflicted individuals will have a really long (1.35 kb) fragment
         - carriers will show all three fragments
- carrier detection of cystic fibrosis
   - with the ΔF508 mutation: allele-specific oligonucleotide (ASO) detection
      - radiolabeled ASO complementary to the normal sequence is used to make a probe
      - radiolabeled ASO complementary to the mutant sequence is used to make another probe
      - DNA (often PCR-amplified) is spotted on both probes
      - carrier, normal, or afflicted is determined by where the fluorescent spot occurs
   - without the ΔF508 mutation: RFLP linkage analysis
      - an RFLP that is tightly linked to the mutation is found
      - DNA from the family is digested, and the inheritance to an afflicted child is analyzed
      - from this, it can sometimes be deduced whether or not a given unafflicted child is a carrier
- disease
   - sickle cell anemia:
      - cause: E6V (glutamate to valine) mutation in the β-globin allele
      - effects: asymptomatic carriers; anemia and associated symptoms in afflicted
   - cystic fibrosis
      - cause: any of numerous mutations in the cystic fibrosis transmembrane regulator (CFTR)
      - effects: defective Cl- transport; serious effects on respiration and digestion, clogged pancreas, death ~25
you should understand:
- RFLP markers
   - RFLP analysis is based on the idea that tightly-linked traits will segregate together during meiosis
      - the RFLP trait and the disease trait are not causally linked!
      - presence of RFLP can only be used to deduce the segregation of disease traits
   - whether an RFLP is present or not in a diseased chromosome is a matter of luck
                                                                       Biochemistry: NOTES & OBJECTIVES (page 23 of 165)



    - in parents where RFLP is present on one homolog, given an afflicted child, carrier status can be determined
    - in parents where either parent does or does not have the marker in both homologues, deduction is difficult


Notes: Lecture and Reading
general considerations for genetic testing
- testing vs. screening
   - genetic test: done in individuals considered likely to bear the diseased allele
   - genetic screening: application of a genetic test to a large population
      - most genetic screening is too expensive to justify wide application
      - instead, screening is typically limited to populations known to be at risk
- goals of genetic testing
   - identify adult carriers of debilitating or fatal diseases in order to guide reproductive choices
   - identify fetuses that will develop such diseases in order to guide termination decisions
   - identify inborn disorders that require prompt treatment or prophylactic measures
- adults vs. newborns
   - adults: relatively straightforward due to availability of tissue
   - fetuses: difficult due to the small amounts of tissue able to be obtained from a fetus
direct detection of a mutation: sickle cell anemia
- sickle cell anemia: overview
   - autosomal recessive disorder caused by a E6V mutation in β-globin
   - HbS: formed of α2βS2
      - aggregates when deoxygenated, forms long fibers
      - this alters cellular shape, causing cells to get stuck in capillaries, and leading to tissue damage
   - sickle cells are prone to lysis and last a few weeks in blood rather than the usual 4 months
- identification of adult carriers: microscopic and electrophoretic examination
   - microscopic examination: some βAβS heterozygote RBCs in low O2 will result in sickle cell shape
   - electrophoretic examination: HbS and HbA in a carrier can be identified due to G, V charge differences
- identification of fetal carriers: restriction enzyme analysis
   - blood cannot be drawn, so a DNA test must be used
   - mutation is A to T in the non-template strand that destroys a MstII restriction enzyme recognition site
      - flanking cleavage sites: 1150 bp and 200 bp away
      - βS allele: 1.35 kb fragment
      - βA allele: 1.15 kb, 0.2 kb fragments
   - Southern blot analysis can be used to detect the fragments in a digest of DNA extracted from fetal tissue
   - note: MstII site destruction could be due to other mutations; this test thus assesses presence or absence of βA
- identification by sequence: allele-specific oligonucleotides (ASO)
   - theory
      - ASO, matching the normal DNA sequence and encompassing site of mutation, is synthesized chemically
      - under appropriate conditions of temperature and [salt], ASO should hybridize only to the normal sequence
      - can be used to detect any specific sequence change
   - practice
      - DNA from a tissue sample is spotted onto a membrane and incubated with:
         - strip 1: radiolabeled ASO complementary to normal sequence
         - strip 2: radiolabeled ASO complementary to mutant sequence
      - spots will glow based on which sequence is present
      - note that PCR is often used to amplify the chromosomal segment containing the sequence in question
inference of a mutation by linkage to an RFLP or SNP: cystic fibrosis
- restriction fragment length polymorphisms: an overview
   - precise identity of a mutation must be known in order for ASO analysis to work
      - sometimes the defective gene has not been identified
      - sometimes many different mutant alleles exist within a given population
   - in such cases, inheritance can sometimes be traced using a linked genetic marker
   - RFLP: natural variations in DNA; no effect on phenotype, but can be detected by restriction enzyme digestion
- cystic fibrosis: an overview
   - autosomal recessive disorder caused by mutation in cystic fibrosis transmembrane regulator (CFTR)
                                                                       Biochemistry: NOTES & OBJECTIVES (page 24 of 165)



      - CFTR: regulates transport of Cl- ions across cellmembranes
      - symptoms usually include respiratory and digestive problems
         - lungs become clogged with mucous and are susceptible to pneumonia
         - pancreatic duct becomes clogged, and digestive enzymes fail to reach the intestines
      - 1/2000 U.S. newborns is afflicted
      - median survival age for individuals with CF is about 25 YO
- diagnosis
   - CF: causes excess salt in sweat
   - CF carriers: no detectable phenotype, so a DNA test is required
- mutations
   - ΔF508: 70% of carriers; deletion of phenylalanine at position 508
      - blocks protein’s transit from ER to cell membrane, thus blocking its function in Cl- transport
      - can be detected with ASO
   - other 30% of carriers: more than 200 causative mutations, making ASO much more difficult
- linkage analysis
   - situation 1
      - parents: disease alleles on chromosomes with site, normal alleles on chromosomes lacking site
      - children: homozygous uncut = normal, heterozygous = carrier, homozygous cut = afflicted
   - situation 2
      - parents: disease alleles on chromosomes with site, one parent’s normal allele also has site
      - children: homozygous cut = afflicted OR carrier, heterozygous = carrier OR normal
   - situation 3
      - parents: one parent has disease allele with site and normal without, other parent has opposite
      - children: homozygous cut/uncut = carrier, heterozygous = afflicted OR normal
   - situation 4
      - parents: both parents have restriction sites on all four chromosomes
      - children: all will have restriction sites, and thus this site is not useful
   - there is no obligatory relationship between RFLP and a disease




17. Transcriptional Control of Gene Expression
Study Guide
be able to:
- eukaryotic protein-coding gene
   - upstream elements
      - upstream promoter elements: within a few hundred base pairs upstream of initiation site
      - TATA box: about 30 bp upstream of the initiation site
   - transcription start site: found shortly after TATA box
   - exons and introns: found downstream of the transcription start site
      - always an odd number of exons
      - always an even number of introns
   - enhancer locations: thousands of base pairs away in an orientation-independent manner
      - found within introns
      - found far upstream or downstream of the gene
- steroid hormone mechanism of gene transcription regulation
   - steroid hormones: cholesterol-derived molecular signal
   - enter target tissue by diffusion through plasma membrane and bind their nuclear receptor
   - binding of hormone releases Hsp90, allowing hormone/receptor complex to bind DNA
   - this binding regulates (usually promotes) binding of RNAp, along with other factors (TFIIB, TFIID)
- activation of PEPCK
   - cortisol: adrenal steroid hormone; binds glucocorticoid receptor, which binds GRE and promotes PEPCK
   - glucagon: polypeptide hormone that signals low blood glucose, promoting PEPCK
                                                                       Biochemistry: NOTES & OBJECTIVES (page 25 of 165)



   - adrenaline: adrenal hormone that signals need for glucose, promoting PEPCK
      - both cause an upregulation of cAMP, which binds CREB, and complex binds CRE
you should know:
- combinatorial control
   - numerous genes may come together to repress or (more commonly) activate gene transcription
   - it is the combined effect of all elements that determines the total regulation
- mechanism of Jun/Fos promoters
   - α-helices containing a leucine zipper and a basic region
      - hydrophobic leucines at every 7th residue face the same side, come together
      - (+) charged basic regions oriented to fit into the grooves of DNA, where they interact with (-) charged DNA
   - bind at the AP-1 promoter element
   - dimerization
      - Jun/Jun: bind poorly
      - Jun/Fos: bind extremely well
      - Fos/Fos: do not bind at all
you should understand:
- regulation
   - general transcription factor: trans-acting elements required for transcription of all protein-coding genes
   - gene regulatory protein: modify basal level of transcription by TFs, in a gene-specific manner
- domains of a transcriptional activator protein
   - DNA-binding: specifically interacts with and binds DNA, about 8-10 bp long
   - activation: interacts with general transcription factors
   - effector: alters ability to activate transcription in response to a cellular signal


Notes: Lecture and Reading
overview of regulation of gene expression
- cellular identity
   - multicellular organisms must coordinate levels of gene expression
   - intercellular signals: hormones, growth factors, cell to cell contact, amongst others
- controlling gene expression
   - transcriptional control (most common)
   - processing control
   - translational control
   - degradation control
- components of gene expression
   - factors: proteins, RNA, or complexes thereof that act on signals or elements present in DNA, RNA, or protein
   - promoter elements: DNA sequences near the gene that aid in the binding of RNAp II
   - cis-acting elements: act on a local scale, with limited expression
      - example: sequence elements
      - inherited defects in gene expression tend to be caused by mutation of cis-acting elements
   - trans-acting elements: act on a global scale, across numerous molecules
      - example: transcription factors
      - mutations in transcription factors are typically lethal very early on, and are often not recognized
control of gene expression: DNA sequence elements
- overview
   - DNA transcription level is generally controlled by the interaction of trans-acting and cis-acting elements
   - cis-acting sequence elements: collectively termed “promoter”
   - trans-acting elements
      - general transcription factors (TFs): required for transcription of protein coding genes
      - gene regulatory proteins: modify basal level of transcription directed by TFs in a gene-specific manner
         - activators: increase transcription
         - repressors: decrease transcription
- RNAp II: initiation of transcription
   - general transcription factors
      - TFIID: binds sequence 5’-TATAAA-3’ (TATA box) at ~30 base pairs upstream of transcription start site
                                                                           Biochemistry: NOTES & OBJECTIVES (page 26 of 165)



      - TFIIB: binds adjacent to TFIID
   - transcription initiation: process
      - TFIID and TFIIB bind to DNA, often joined by other factors (such as TFIIA)
      - RNAp II recognizes the DNA complex, binds, and begins transcription
      - TFIID and TFIIB stay bound to the promoter after initiation, promoting additional recruitment of RNAp II
      - process requires several activator proteins
- activators in human gene expression
   - activator binding sites
      - upstream elements: binding sites for activator proteins just upstream of the promoter
      - enhancers: binding sites located thousands of base pairs away; orientation-independent
   - activator proteins
      - DNA-binding domain: recognizes a specific DNA sequence 8-10 bp long
      - activation domain: interacts with general transcription factors
      - effector domain: interacts with a cellular signal (e.g. hormone, phosphorylation)
         - found only in certain activator proteins
         - other gene regulatory proteins are always on, thus activity is determined primarily by their concentration
- combinatorial control: phosphoenolpyruvate carboxykinase (PEPCK)
   - definitions
      - combinatorial control: level of synthesis determined by net effect of all bound regulators
      - PEPCK: key role in gluconeogenesis; produced primarily in the liver
   - PEPCK structure
      - TATA box:                        -30
      - CRE:                             -100
      - AP-1 promoter:                   -125, -250, -275
      - GRE:                             -360
      - HNF4α binding site:              -400
   - receptors
      - cyclic AMP response element (CRE)
         - glucagon and adrenaline (which signal need for glycolysis) stimulate production of cAMP
         - cAMP stimulates a protein kinase that activates the protein CREB
         - cAMP binds CREB, complex binds CRE, promoting transcription
      - AP-1 promoter: bind Jun/Fos general activators, regulated in some part by their synthesis
      - glucocorticoid response element (GRE)
         - DNA sequence element
         - binds hormone/glucocorticoid receptor (GR) complex, which increases transcription
      - hepatocyte nuclear factor 4α (HNF4α)
         - tissue-specific activator, present primarily in the kidney and liver
         - absence of this factor restricts synthesis in other tissues, even if cortisol, glucagon, or adrenaline is high
nuclear receptors
- nuclear receptors: overview
   - nuclear receptors: gene regulatory proteins that bind small, hydrophobic molecules in their effector domains
   - steroid hormone receptors: have a steroid-derived hormone receptor, such as cortisol, estrogens, or androgens
      - steroids are lipid soluble, and can thus diffuse through cell membrane to bind a nuclear receptor
      - this allows direct action, as opposed to the indirect use of second messengers such as cAMP
   - other examples of molecules using nuclear receptors
      - thyroxine (thyroid hormone), vitamin D, retinoic acid (derived from vitamin A)
      - ligand for these is currently unknown
- nuclear receptor structure
   - structure
      - variable N-terminal receptor (transcription activator)
      - DNA-binding domain
      - C-terminal ligand-binding domain
   - ligand binding
      - Hsp90: inhibitory protein that complexes nuclear receptors without bound ligand
      - upon binding of ligand, Hsp90 is released, and complex can bind to DNA to regulate transcription
- combinatorial control: HNF4α
                                                                       Biochemistry: NOTES & OBJECTIVES (page 27 of 165)



   - DNA binding: the effect of mutation
      - amino acids of DNA-binding domain of protein make highly specific contacts with DNA bases
      - this allows the domains to precisely read the sequence
      - mutation of even a single base pair can significantly disrupt this contact
   - Factor IX
      - overlapping receptors
         - androgen receptor: binds testosterone to activate transcription
         - HNF4α: orphan nuclear receptor and tissue-specific activator
      - mutations in Factor IX
         - Leyden mutation
            - occurs at -20, impacting the HNF4α binding site
            - this causes hemophilia in young children, but males improve after puberty due to androgen receptor
         - Brandenburg mutation
            - occurs at -26, disrupting both the HNF4α and androgen receptor binding sites
            - this causes lifelong hemophelia, as both binding sites are disrupted
   - HNF4α in the kidney and pancreas: effects of mutation
      - maturity-onset diabetes of the young, type 1 (MODY1): rare autosomal dominant; caused by mutation
      - type 2 diabetes: increased risk based on single nucleotide polymorphisms in HNF4α
   - underscores the importance of HNF4α in sugar metabolism
Jun and Fos: leucine zipper
- structure
   - long α-helices with two domains
      - leucine zipper: Leu side chains at every seventh position, forming a hydrophobic stripe on one side
      - basic region: positively charged, can interact with negatively-charged DNA
   - two of these helices come together, forming a dimer stabilized by hydrophobic Leu contacts
   - after dimerization, basic region contacts DNA
- activation
   - heterodimers vs. homodimers
      - Jun/Jun: bind AP-1 site to some degree
      - Fos/Fos: do not bind DNA
      - Jun/Fos: bind DNA better than Jun homodimers
   - Fos
      - increases transcriptional activation by Jun
      - stimulated by growth factors; may help initiate cell division
      - overexpression: can cause cancer




18. Protein Synthesis
Study Guide
do the following:
- structures
   - aminoacyl AMP: AMP with amino acid attached (via carboxyphosphate linkage) to 5’ C
   - aa-tRNA: amino acid attached (via esterification) to 3’ C or 2’C of tRNA N-terminal adenylate residue
- structure of a translating ribosome
   - large (60S) and small (40S) subunit
      - 40S subunit: decoding of mRNA, directly on mRNA
      - 60S subunit: carries out peptidyl transferase reaction
   - ribosomal sites
      - A site: aminoacyl tRNA site, containing the incoming peptide; 3’-most structure
      - P site: peptidyl tRNA site, containing the growing polypeptide chain; middle site
      - E site: exit site, where empty tRNA molecules leave the ribosome; 5’-most structure
   - components
                                                                        Biochemistry: NOTES & OBJECTIVES (page 28 of 165)



      - aminoacyl tRNA: located in A site, contains amino acid to be added
      - peptidyl tRNA: located in T site, contains growing polypeptide chain (N-terminus distal to ribosome)
      - codon: three letter code located on DNA
      - anticodon: complementary three letter code on RNA
      - peptidyl-transferase site: located between ends of the aminoacyl and peptidyl tRNAs
you should know:
- components of translation initiation
   - start codon: AUG (methionine)
   - tRNA: methionyl-tRNAMet(i) (Meti is specific to initiation)
- recognition of the start codon: ribosome looks for first AUG sequence downstream of the 5’-mGppp cap
- EF-1α and protein elongation
   - function: binds GTP, binds an aminoacyl-tRNA, and brings it to the aminoacyl site in the ribosome
   - molecular clock
      - peptidyl transferase can only work after EF-1α has left the site, which requires hydrolysis of GTP
      - binding of aa-tRNA anticodon signals hydrolysis of GTP
   - proofreading: if anticodon does not match, tRNA will dissociate before GTP hydrolysis is complete
you should understand:
- diphtheria toxin
   - EF-2 is a GTP-binding protein that is required for translocation of peptidyl tRNA from A site to P site
   - diphtheria toxin ADP-ribosylates (from NAD+) a specific amino acid residue in EF-2, inactivating it
   - one molecule of toxin is potent enough to kill an entire cell
- tRNAs and the genetic code
   - there are 20 amino acids, 20 aminoacyl-tRNA synthetases and 64 possible amino acid codons
   - redundancy: multiple codons must be recognized by a single aminoacyl-tRNA synthetase
   - as such, many synthetases must use structural features other than tRNA codon in order to bind


Notes: Lecture and Reading
tRNA activation: aminoacylation
- components
   - definitions
      - aminoacyl-tRNA synthetase: enzyme that activates tRNA by attaching amino acid
      - tRNAamino acid: recognizes RNA codon, involved in transferring it to a growing polypeptide
      - anticodon: sequence by which an activated tRNA recognizes and binds DNA
   - there are 20 AA-tRNA synthetases, one for each amino acid
      - more than 20 tRNA molecules required for all codons (some tRNA can recognize multiple anticodons)
      - some synthetases must therefore recognize multiple tRNA molecules
   - isoacceptors: tRNAs that have different anticodon sequences but become charged with the same amino acid
      - some synthetases recognize tRNA molecules by their anticodon sequence
      - synthetases that charge multiple tRNAs must recognize other structural features of the tRNA
- enzymatic process: two steps, both catalyzed by aminoacyl-tRNA synthetase
   - adenylation
      - amino acid + ATP  aminoacyl-AMP + PPi
      - this reaction activates the amino acid for use in the next step
      - fidelity
         - this reaction gives the synthetase another opportunity to proofread the amino acid, increasing fidelity 100X
         - if aminoacyl-AMP does not fit properly, adenylate is hydrolyzed and amino acid is discarded
   - aminoacylation
      - aminoacyl-AMP + tRNA  aminoacyl-tRNA + AMP
      - 2’ or 3’ OH of terminal adenine in tRNA attacks the carboxyphosphate bond formed in adenylation reaction
         - this attaches the amino acid to the tRNA, activating it for use in polypeptide elongation
         - note that every tRNA has an adenylate residue on the 3’ end
- net energy used: 2 phosphates
   - PPi generated in aminoacylation step is hydrolyzed to 2P i by pyrophosphatase
   - this makes the net reaction more exothermic, driving it forward by mass action
ribosome structure and function
                                                                        Biochemistry: NOTES & OBJECTIVES (page 29 of 165)



- definitions
   - peptidyl transferase: enzyme that transfers amino acids from aa-tRNA to the growing polypeptide chain
   - decoding: interaction of tRNA with mRNA wherein tRNA anticodons read RNA codons and add amino acids
- structure
   - large subunit (60S) and small subunit (40S)
      - 40S subunit: decoding of mRNA
      - 60S subunit: carries out peptidyl transferase reaction
   - three sites
      - A site: aminoacyl tRNA site, containing the incoming peptide
      - P site: peptidyl tRNA site, containing the growing polypeptide chain
      - E site: exit site, where empty tRNA molecules leave the ribosome
- the process of translation
   - initiation
      - 40S and 60S subunits are brought together at the first codon to be translated, forming the A, P, and E sites
      - initiation codon: AUG (methionine)
         - in rare cases, codon is GUG or UUG, but methionine is still incorporated
         - in bacteria, the AUG used is somewhat variable, depending on the sequence context
         - in eukaryotes, the first AUG downstream of the 5’ cap is almost always used for initiation
      - initiator tRNA: methionyl-tRNAMet (tRNAMet(i))
         - this is a special methylated form of methionine that is used specifically to initiate transcription
         - binds directly in the P site, rather than the A site
         - often cleaved off later by an N-terminal protease
   - elongation
      - aa-tRNAaa binds in the A site of the ribosome, immediately downstream of the previous codon
      - peptidyl transferase activity of 60S subunit
         - catalyzes attack of A-site free amino group to the P-site tRNA-amino acid ester linkage
         - this displaces the growing polypeptids chain from the P-site to the A-site, leaving the P-site empty
         - reaction is favorable because the aminoacyl linkage has higher energy than the nascent peptide bond
         - note: the catalysis is performed by the RNA of the large subunit, NOT the protein component
      - translocation
         - ribosome moves three nucleotides downstream on the mRNA, ejecting the uncharged tRNA from the E-site
         - this moves the peptidyl-tRNA to the P-site, opening up the A site for the next aminoacyl-tRNA
      - repetition: moves ribosome in 5’3’ direction, with concurrent synthesis of polypeptide in N to C direction
   - termination
      - UAA, UAG, UGA are not recognized by tRNAs, but instead by protein release factors
      - directs ribosome to stop synthesis, and peptidyl transferase activity hydrolyzes the last bond to the tRNA
      - ribosome and the novel protein are released
- polysome: mRNA with several attached, simultaneously-translating ribosomes
   - ribosomes near 5’ end: polypeptide is short, incomplete
   - ribosomes near 3’ end: polypeptide is longer, closer to complete
accessory factors in translation: EF-1α and EF-2
- G proteins
   - G proteins: GTP-binding required for activity
   - both EF-1α and EF-2 are G proteins
   - G proteins used in numerous cellular processes, such as vesicle transport, protein and RNA transport, cell signals
- EF-1α
   - EF-1α: GTP-regulated protein that binds aminoacyl-tRNA and delivers it to the A site
      - upon binding of tRNA anticodon to mRNA codon, GTP is hydrolyzed to GDP + P i
      - after hydrolysis, EF-1α-GDP is released from the ribosome, and peptide bond formation can occur
   - because this process takes time, GTP is acting as a molecular clock
      - peptide bond formation cannot occur until GTP is hydrolyzed and the complex has left the site
      - incorrect AA-tRNA molecules, which bind the codon more weakly, usually dissociate before hydrolysis
      - correct AA-tRNA molecules will remain until hydrolysis occurs
      - this is thus a proofreading step that, along with aa-tRNA synthetase, reduces translational error to 1/10,000
- EF-2
   - EF-2: GTP-regulated protein required for translocation of peptidyl-tRNA from A site to P site
                                                                        Biochemistry: NOTES & OBJECTIVES (page 30 of 165)



   - diphtheria
      - diphtheria: toxin in certain strains of Corynebacterium diphtheria
      - acts by catalyzing transfer of ADP-ribose from NAD+ to a specific amino acid in EF-2, inactivating the protein
      - this blocks protein synthesis by halting the translocation step
      - a single molecule of toxin can kill an entire cell (which contains half a million EF-2 molecules)
   - erythromycin
      - erythromycin: antibiotic that binds large subunit RNA in bacterial ribosome, inhibits translocation
      - because this does not affect eukaryotic translocation, it is an effective antibiotic
      - some bacterial strains are emerging that have a resistance to this
energy of protein synthesis
- addition of each amino acid residue to a growing polypeptide chain requires 4 high energy phosphate bonds
   - aminoacyl-tRNA synthetase          amino acid activation       ATP  AMP + PPi  2Pi
   - EF-1α                              molecular clock             GTP  GDP + Pi
   - EF-2                               translocation               GTP  GDP + Pi




19. Protein Targeting
Study Guide
- protein pathways
   - cytosol
      - ribosome: synthesizes protein and releases into cytosol (default path)
      - certain sequences can cause import into the nucleus or mitochondria
   - endoplasmic reticulum
      - ribosome synthesizes protein, and a signal sequence is recognized by SRP
      - SRP arrests translation, binds an SRP membrane receptor, and docks ribosome with translocon
      - protein is extruded into the ER, becoming membrane-bound or soluble based on stop/start sequences
      - KDEL receptors maintain the protein in the ER, returning them from vesicles via retrograde transport
   - lysozome
      - ribosome synthesized protein, and a signal sequence is recognized by SRP
      - SRP arrests translation, binds an SRP receptor, and docks ribosome with translocon
      - protein is extruded into the ER, becoming membrane-bound or soluble based on stop/start sequences
      - phosphomannose in N-linked oligosaccharides targeted by receptors protein for lysosome
   - cell surface
      - ribosome synthesized protein, and a signal sequence is recognized by SRP
      - SRP arrests translation, binds an SRP receptor, and docks ribosome with translocon
      - protein is extruded into the ER, becoming membrane-bound or soluble based on stop/start sequences
      - protein exits trans-Golgi and is brought to the cell surface (default ER path)
- polypeptide fates: signal sequences
   - signal peptidase, no stop transfer:
           soluble protein secreted outside the cell
   - internal uncleaved signal peptidase, no stop transfer:
           IMP on cellular membrane, with N-terminus inside, C-terminus outside cell
   - internal uncleaved signal peptidase, one stop transfer:
           IMP on cellular membrane, with one extracellular loop
- folding assistants
   - Hsp90: molecular chaperone that permits proteins to fold without aggregating
   - protein disulfide isomerase: catalyzes disulfide bond cleavage, allowing proteins to attain lowest energy state
know the following:
- signal recognition particle (SRP)
   - binds ribosomes from which a signal sequence has emerged
   - arrests translation (by blocking the A site) and brings ribosome to ER membrane
   - after binding a receptor, SRP docks the ribosome over a translocon
                                                                        Biochemistry: NOTES & OBJECTIVES (page 31 of 165)



   - SRP leaves, allowing translation to continue into the translocon
- N-linked glycosylation
   - ER-mediated attachment of sugars to asparagine residues of nascent polypeptides
   - because this only happens within the ER, IMPs that are N-glycosylated will only have extracellular sugars
   - recall: extracellular, ER lumen, lysosomal lumen are all topologically equivalent
- I-cell disease
   - mannose phosphokinase, which N-glycosylates certain proteins, is defective
   - phosphomannose receptors target proteins for the lysosome
   - proteins that should be targeted for the lysosome are instead secreted by the default pathway
   - lysosomes are unable to do their job; this leads to severe psychomotor retardation, skeletal defects


Notes: Lecture and Reading
localization signals in protein transport: overview
- signal sequence
   - signal sequence: localization signal that specifies synthesis into lumen of ER
   - usually found at N-terminus of a protein
   - consists of basic amino acid (Lys or Arg) followed by a stretch of hydrophobic residues
- proteins with signal sequence
   - synthesized into ER, where default path is through Golgi apparatus and constitutive secretion on the cell surface
      - some specific signals can cause proteins to be secreted in a regulated fashion from secretory vesicles
      - other specific signals can cause proteins to be sent to the lysosome
   - retention signals for organelle-specific proteins of ER, Golgi allow reuse and recycling of those proteins
   - ER import of proteins is co-transcriptional
- proteins lacking a signal sequence
   - synthesized and secreted into the cytosol, where default path is to remain there
      - nuclear import signals
         - direct proteins to nucleus
         - usually contain several basic residues
      - mitochondrial import signals
         - direct proteins to mitochondria
         - usually amphipathic helices with basic residues on one face, hydrophobic residues on other
   - cytosolic import of proteins is post-transcriptional
the endoplasmic reticulum: entrance to vesicular transport pathway
- protein translation
   - signal recognition particle (SRP): binds ER signal in polypeptides emerging from ribosome
      - consists of RNA and several proteins
      - causes translation to stop, and docks with an SRP receptor located on the membrane
   - translocon: pore through ER membrane
      - when inactive, contains a plug in the ER lumen that prevents free pass of molecules
      - SRP/SRP receptor/ribosome complex binds to translocon
      - translation begins again, and elongating protein is extruded into ER
   - post-translation
      - SRP is released from elongating ribosome, floats away to find another signal peptide
      - ribosome dissociates from translocon, and mRNA becomes soluble again
      - mRNA usually stays attached to the membrane via other ribosomes in the polysome
- protein positioning
   - components
      - stop sequences
         - hydrophobic sequence in polypeptide that signals a stop in translocation
         - this gets stuck in the translocon, and is extruded to become part of the membrane of the ER
      - start sequences
         - hydrophobic sequence in polypeptide that signals the beginning of translocation
         - these are brought to a translocon similar to signal sequences
      - signal peptidase
         - cleaves the signal peptide, creating a new N-terminal end that faces the inner ER lumen
                                                                          Biochemistry: NOTES & OBJECTIVES (page 32 of 165)



         - signal sequence remains in the ER lumen until it is degraded by other enzymes
   - function
      - use of signal peptidases, stop sequences, and start sequences alters the orientation and position of the protein
         - each odd-numbered hydrophobic segment acts as a signal peptide or start transfer sequence
         - each even-numbered hydrophobic segment acts as a stop transfer sequence
      - polarity of integral membrane proteins
         - signal sequence, when cleaved by peptidase, places the N-terminal region within the ER lumen
         - if signal sequence is further into the polypeptide and is not cleaved by peptidase, N-terminus in cytosol
         - knowing this, protein orientation can be predicted
         - because of how vesicles work, the ER lumen is topographically equivalent to extracellular membrane
- proper folding: molecular chaperones
   - binding protein (BiP): molecular chaperone protein present in high concentration in ER lumen
   - molecular chaperone: protein specialized to guide the folding and assembly of other proteins
      - proteins are extruded into the ER lumen in an extended state, and are not properly folded
      - this promotes non-specific aggregation of exposed hydrophobic regions
      - molecular chaperones shield forming polypeptides, giving them time to fold individually
- proper folding: disulfide bonds
   - protein disulfide isomerase (PDI): catalyzes cleavage of disulfide bonds
   - disulfide bonds
      - cytosol: reducing environment (favors removal of disulfide bonds)
      - ER lumen: oxidizing environment (favors addition of disulfide bonds)
   - upon entry into ER, cysteine disulfide bonds form spontaneously, often incorrectly
   - PDI cleaves bonds, allowing protein to continue towards its lowest energy (most stable) state
- protein glycosylation
   - N-linked glycosylation: attachment of sugars to amino group of certain asparagine residues
      - most proteins entering ER are covalently modified in this way
      - glycosyl transferase: catalyzes this process on luminal side of ER membrane
   - only happens within the ER lumen
      - secretory vesicles work such that ER side of integral membrane proteins (IMPs) will face outside cell
      - because of this, only extracellular portions of IMPs are glycosylated
the Golgi apparatus and beyond
- Golgi apparatus structure
   - Golgi apparatus consists of 4-8 cisternae organized in a stacked fashion
   - cisternae: disk-shaped membrane bound vesicles
- movement through the Golgi
   - cis face: cisterna closest to ER (also called transitional ER)
   - trans face: cisterna closest to plasmalemma
   - proteins move stepwise through cisterna in vesicles that bud off each face and merge with the next
   - from the trans face, vesicles move to the lysosome, secretory granules, or directly to the plasmalemma
   - retrograde transport: returns proteins to ER
      - occurs through the use of retrograde vesicles that bud off and return to ER
      - proteins to be returned to ER (e.g. BiP or PDI) are recognized by KDEL receptor
      - KDEL receptor: Lys-Asp-Glu-Leu conserved sequence
- Golgi function
   - localization: proteins are sent to proper places within the cell
      - this often occurs by enzymes binding to specific receptors on Golgi membranes
      - example: lysosome
         - N-linked oligosaccharides of lysosome enzymes are recognized by phosphomannose receptors
         - phosphomannose receptors bind, package enzymes into vesicles bound for lysosome
      - I-cell disease: autosomal recessive disorder characterized by psychomotor and skeletal difficulties
         - defect in kinase that phosphorylates mannose in N-linked oligosaccharides of lysosomal enzymes
         - phosphomannose receptor fails to recognize this, and proteins are secreted instead
         - lysosome is unable to complete its digestion, leading to large cellular inclusions
   - glycosylation
      - N-linked sugars can be trimmed, and different sugars can be added in order to modify oligosaccharides
      - other proteins are glycosylated directly in the Golgi
                                                                       Biochemistry: NOTES & OBJECTIVES (page 33 of 165)



        - O-linked glycosylation: oligosaccharides added to –OH of Ser, Thr residues
        - less prevalent than N-linked glycosylation
     - glycosylation functions: proper folding, stability, and cell-cell interactions
  - cleavage
     - in trans-Golgi and beyond, some proteins are cleaved into mature form
     - example: insulin
        - ribosomal synthesis:                           preproinsulin
        - ER signal peptide removal:                     proinsulin
        - secretory vesicle internal peptide removal: insulin
     - numerous other peptide hormones and neuropeptides are made in a similar fashion




20. Posttranscriptional Control of Gene Expression
Study Guide
do the following:
- absorption and transport of iron
   - divalent metal ion transporter 1 (DMT1) channel protein brings iron into the enterocyte
   - ferroportin channel protein secretes iron into the bloodstream
   - apotransferrin (Tf) binds plasma iron, becoming transferrin, and brings it to a target cell
   - transferrin receptor (TfR) on the target cell binds and internalizes Tf within endosomes (cellular vesicles)
   - acidic endosomes cause release of iron, which enters cytosol through DMT1
   - apotransferrin and TfR are returned to plasma membrane and reused
   - ferritin stores any excess Fe
- regulation of ferritin, transferrin
   - iron response element (IRE): promoter loop found in 5’ end of ferrritin, 3’ end of TfR
   - iron regulatory protein 1 (IRP1): cytoplasmic aconitase without bound iron; binds and masks IRE
   - high [Fe]
      - IRP1  cytoplasmic aconitase, leaving IREs in mRNA unoccupied
         - ferritin: exposed IRE at 5’ end promotes translation initiation, leading to higher [ferritin]
         - transferrin receptor: exposed IREs at 3’ end promote degradation, leading to lower [TfR]
      - enterocytes: promotes uptake of iron and loss to feces as enterocytes are sloughed
      - other cells: promotes iron storage, limits further uptake of iron, decreasing intracellular [Fe]
   - low [Fe]
      - cytoplasmic aconitase  IRP1, which binds IREs in mRNA
         - ferritin: blocks initiation of translation, leading to lower [ferritin]
         - transferrin receptor: blocks 3’ end and exonuclear degradation, leading to higher [TfR]
      - enterocytes: iron capture is limited, shunting iron into the bloodstream
      - other cells: low ferritin and high TfR increase uptake and intracellular [Fe]
you should know:
- splice site recognition: U1 of spliceosome, finding a 5’-GUAAGU-3’ mRNA sequence (GU most important)
- forming different sizes of apolipoprotein B
   - B100 (liver): full protein is translated
   - B-48 (intestine): post-transcriptional deamination of cytosine (forming uracil) forms a premature stop codon
- stability of Fos
   - destabilization elements found in coding region, 3’-UTR region
   - both lead to more rapid 3’ exonuclease activity
   - this is critical, as overexpression of Fos is linked to cancer
- iron: primarily used for oxygen transport
you should understand:
- alternative splicing
   - mutually exclusive exons
      - differential use of splicing sites, as in smooth/striated muscle
                                                                           Biochemistry: NOTES & OBJECTIVES (page 34 of 165)



     - allows spliceosome to skip or include certain exons
  - alternative 3’ terminal exons
     - use of optional intron containing a poly(A) site, as in Ig heavy chains
     - if the optional intron is used, the first poly(A) site is where translation ends
     - if the optional intron is not used, the terminal poly(A) site is used


Notes: Lecture, Reading
advantages of posttranscriptional control of gene expression
- multiple proteins from one gene
   - happens through alternative splicing, RNA editing
   - increases repertoire of proteins that can be made from a fixed number of genes
   - 1/3 to 1/2 of all human genes produce pre-mRNAs that are subject to alternative splicing
- faster regulation
   - transcriptional regulation is on the order of hours because of the amount of time required
   - rapid modulation can occur at the level of the mature mRNA through changes in stability, translation efficiency
- takeover during quiescent transcription
   - at some stages in life (e.g. embryogenesis), translation does not occur
   - it is still important for proteins present to be accountable to regulation
alternative splicing: mechanism and examples
- mechanism of splicing
   - splice site: intron/exon junctions that are the site of splicing
      - 5’ splice site: GU followed by a number of preferred nucleotides
      - 3’ splice site: AG preceded by a polypyrimidine (U, C) rich sequence
      - deviations from these sequences increase likelihood of overlooking splice site
   - U1 snRNA: one of 5 snRNAs required for splicing
      - U1 snRNP recognizes and binds 5’ splice site
      - protein splicing factors recognize, bind 3’ splice site
   - reaction
      - factors excise the intron, attach 5’ splice site to an internal branch point, making a lariat shaped structure
      - lariat is degraded by nucleases
- alternative splicing
   - alternative splicing: use of different splicing sites to modify identity of introns and exons
   - mutually exclusive exons: α-tropomyosin (α-TM)
      - α-tropomyosin: regulatory protein in muscle contraction, blocking binding of actin to myosin
      - alternative splicing creates a slightly different molecule, based on tissue location
         - smooth muscle: exon 3 is skipped, and only 1, 2, and 4 are included
         - striated muscle: exon 2 is skipped, and only 1, 3, and 4 are included
      - mechanism: tissue-specific splicing regulatory factors bind and mask alternative splice sites
   - alternative 3’-terminal exons: immunoglobin (Ig)
      - immune response: causes antibodies to change from membrane-bound to soluble forms
      - alternative terminal exons in the Ig change the C-terminus from hydrophobic to hydrophilic
         - membrane-bound
            - optional intron is spliced out
            - terminal exon is hydrophobic, with poly(A) site 2
         - soluble
            - optional intron remains, and an internal poly(A) site (1) causes premature termination
            - terminal exon is hydrophilic
      - like with mutually-exclusive exons, tissue- or stage-specific factors modify the splicing
RNA editing: mechanism and example
- mechanism
   - RNA editing: post-transcriptional insertion, deletion, or adjustment of individual nucleotides
   - much less common form of RNA processing
- examples
   - apolipoprotein B: protein involved in fat transport
      - intestinal mucosa: produces a 240 kD protein
                                                                          Biochemistry: NOTES & OBJECTIVES (page 35 of 165)



      - liver: produces a 500 kD protein
         - in the intestine, a cytosine base is deaminated, converting it to uracil
         - this results in a stop codon and truncation of the intestinal protein
      - APOBEC-1: enzyme that edits apo B mRNA
         - related enzyme, APOBEC-3G, defends cells against viruses by heavily modifying their DNA
         - unfortunately, HIV produces a protein (Vif) that protects the virus from this
   - AMPA receptors: glutamate receptors required for CNS function
      - two sites are edited, causing deamination of adenosine to inosine (guanine analog)
      - both result in amino acid substitution
      - one is critical, as mice unable to edit the site develop seizures and die by 3 weeks
      - editing at the other site increases with a developing brain, and may influence neuron function
RNA stability: changing the rate of protein synthesis
- overview of mRNA stability
   - rate of mRNA degradation is a means of controlling protein levels
   - mRNA stabilities vary greatly, with half lives from less than 15 minutes to more than 10 hours
      - default state: long half life
      - unstable mRNA: contains cis-acting elements that direct their rapid degradation
- removal of poly(A) tail
   - stabilizing factors: increase average length of poly(A) tails
      - by default, 3’ exonucleases progressively shorten the poly(A) tail of mRNAs
      - degradation from ~200 to <30 leads to rapid degradation of the mRNA
      - poly(A) binding protein (PABP): binds ~30 nucleotides, protecting from degradation
   - destabilizing factors: lead to rapid degradation of poly(A) tail
      - overexpression of Fos can be dangerous due to propensity for forming tumors
      - Fos contains sequences in the coding region and the 3’-UTR that lead to rapid degradation
      - either sequence, when inserted into more stable proteins like β-globin, dramatically reduce half lives
      - mechanism is currently unknown
- micro RNA (miRNA) regulation
   - miRNA: 22 nt RNA sequences complementary to sequences in 3’-UTR of target mRNAs
   - bound micro RNA directs endonuclease cleavage of the protein, allowing exonucleases to degrade the mRNA
   - mechanism and commonality is still poorly characterized
posttranscriptional regulation of iron uptake into the body
- overview: the importance of iron
   - function: required for hemoglobin, myoglobin, cytochromes, certain non-heme enzymes
   - storage: 2-3 g present in protein, and an additional reserve of 1g is stored in the body
   - iron loss
      - 1 mg of iron is lost per day in a healthy adult male
      - 0.5 mg iron lost in addition in menstrual losses in females
      - daily loss even greater during gestation (transfer to fetus)
   - iron absorption
      - absorbed as free iron or as heme
      - absorbed poorly (1/8 of dietary iron is actually retained)
- iron absorption: enterocytes
   - components of iron regulation
      - divalent metal transporter 1 (DMT1): apical channel protein that takes in Fe2+
      - ferritin: large, multisubunit protein that can act as a store of iron in enterocytes, thus regulating iron intake
      - apoferritin: ferritin without bound iron (capacity of about 4500 iron ions
      - ferroportin: basal channel protein that passes iron to bloodstream
   - mechanism of iron regulation
      - high iron: increased enterocyte apoferritin synthesis, trapping iron in enterocytes that are eventually sloughed
      - low iron: repressed apoferritin synthesis, allowing iron to pass into the bloodstream
- iron transport in the bloodstream: transferrin
   - components of iron transport
      - transferrin (Tf): binds plasma iron tightly at physiological pH
      - transferrin receptor (TfR): cell-surface receptor that allows internalization of transferrin
   - mechanism of iron transport
                                                                      Biochemistry: NOTES & OBJECTIVES (page 36 of 165)



      - Tf binds iron at physiological pH, transports it throughout the bloodstream
      - a cell with TfR complexes with Tf, internalizes it within endosomes
      - acidic environment leads to release of Fe, which enters cytosol through DMT1
      - apotransferrin and receptor are returned to the plasma membrane for reuse
      - much of iron is taken in by mitochondria for use in heme synthesis and other purposes
      - excess cytoplasmic iron: stored in ferritin
- regulation of trasferrin receptor and ferritin: iron responsive element (IRE)
   - components
      - IRE: cis-acting stem loop RNA structure found in mRNA of both transferrin and ferritin
         - ferritin: a single IRE in 5’ UTR
         - transferrin receptor: several IREs in 3’ UTR
      - iron regulatory protein 1 (IRP1): trans-acting intracellular iron concentration sensor
         - identical to cytoplasmic aconitase, except that IRP1 lacks 4 iron atoms for activity
         - thus the protein is actually IRP1/cytoplasmic aconitase
   - mechanisms
      - high [Fe]
         - IRP1  cytoplasmic aconitase, leaving IREs in mRNA unoccupied
         - ferritin: exposed IRE at 5’ end promotes translation initiation, leading to higher [ferritin]
         - transferrin receptor: exposed IREs at 3’ end promote degradation, leading to lower [TfR]
         - enterocytes: promotes uptake of iron and loss to feces as enterocytes are sloughed
         - other cells: promotes iron storage, limits further uptake of iron, decreasing intracellular [Fe]
      - low [Fe]
         - cytoplasmic aconitase  IRP1, which binds IREs in mRNA
         - ferritin: blocks initiation of translation, leading to lower [ferritin]
         - transferrin receptor: blocks 3’ end and exonuclear degradation, leading to higher [TfR]
         - enterocytes: iron capture is limited, shunting iron into the bloodstream
         - other cells: low ferritin and high TfR increase uptake, intracellular [Fe]
- iron overload
   - iron overload: accumulation of too much iron in the body
      - at diagnosis, patients often have >10 g iron in the body, sometimes as much as 50 g
      - serum transferrin is >50% saturated (compared to 30% for normal)
   - hereditary hemochromatosis (HH): most common form of inherited disease
      - causative mutation in HFE gene, but the function of that protein is not well understood
      - homozygotes are relatively common (1/200)
      - symptoms: at middle age; including cirrhosis, bronze pigmentation, diabetes mellitus, cardiomyopathy
      - treatment: weekly bleedings
   - iron overload as a complication of sickle cell anemia, thalassemia major
      - intestinal absorption of iron increased as the body attempts to make more RBCs
      - blood transfusions bring even more iron in
      - treatment: chelating agents (e.g. deferoxamine) that bind iron, promote its disposal in the urine




21. Posttranscriptional Control of Gene Expression
Study Guide
be able to:
- mutation-inducing radiation
   - ionizing radiation (e.g. X-rays)
      - initial effect: cause double-stranded break in DNA
      - mutations: DNA rearrangement such as inversions, translocations
   - ultraviolet (UV) radiation
      - initial effect: dimerization of adjacent pyrimidine bases, especially T=T
      - mutations: require DNA repair processes that can sometimes be error-prone
- nucleotide excision repair pathway
                                                                      Biochemistry: NOTES & OBJECTIVES (page 37 of 165)



   - target: chemically-modified DNA, such as T=T dimers
   - mechanism
      - proteins recognize, bind DNA
      - endonucleases make 5’, 3’ incisions in the damaged strand
      - helicase removes damaged strand (to be digested by nucleases)
      - common repair pathway seals DNA, using undamaged strand as a template
   - defect consequences: xeroderma pigmentosum, which includes lesioning and cancers in the skin
- mismatch repair pathway
   - target: mismatched bases or slippage loops
   - mechanism
      - proteins recognize newly-synthesized strand, bind
      - endonucleases make 5’, 3’ cuts
      - exonucleases directly digest DNA
      - common repair pathway seals DNA, using single strand template
you should understand:
- inherited cancer defects
   - proto-oncogenes: accelerate replication and cell growth, oncogenes inherited dominantly
   - tumor suppressor genes: inhibit replication and cell growth; defects inherited recessively
- xeroderma pigmentosum
   - inherited defect in the nucleotide excision pathway
      - A, C: defect in recognition (pyrimidine dimer binding)
      - F, G: defect in incision (exonucleases)
      - B, D: defect in excision (helicases)
      - V: defect in error-free bypass
   - defects in any of these increase likelihood of use of error-prone bypass
   - in error-prone bypass, defects are repaired essentially at random, causing numerous mutations
- MSH2 gene
   - codes for a gene in mismatch repair
      - inheritance of repair defect: autosomal recessive
      - inheritance of predisposition: autosomal dominant
   - receiving one bad copy increases likelihood that other will be knocked out, yielding cancer


Notes: Lecture and Reading
DNA damage and mutations
- DNA damage is usually repaired prior to replication
- consequences of replicating damaged DNA
   - cell death: DNAp stops, synthesis is arrested, and cell undergoes apoptosis
   - error-free bypass: DNAp falls off, specialized high fidelity DNAp repairs mutation, and replication resumes
   - error-prone bypass: specialized DNAp is error-prone, inserts nucleotides at random, leading to mutations
- error-prone bypass is the most carcinogenic
mechanisms of DNA damage
- deamination: removal of an amine group (frequently on C)
   - deaminated bases: altered pairing properties
   - deamination of C gives T, which pairs with A instead of G
- depurination: removal of a purine base by spontaneous hydrolysis of the glycosidic linkage (frequently G)
   - alkylating agents: induce depurination (examples: some chemotherapeutic drugs)
   - Rev1: inserts dCMP opposite a missing base in the template strand
- dimerization: covalent linkage of adjacent pyrimidine bases (especially T=T)
   - caused by ultraviolet (UV) radiation
   - stop normal replicative polymerase, can be bypassed by a specialized DNAp
- double-stranded breaks: separation of DNA across both strands
   - caused by X-ray radiation
   - leads to highly reactive free ends of DNA
      - inversions: flipping orientation of DNA without changing position
      - translocations: fusion of DNA from two different chromosomes
                                                                        Biochemistry: NOTES & OBJECTIVES (page 38 of 165)



common features of DNA repair pathways
- 99.9 % of DNA damage is repaired prior to mutation
- common pathway of repair
   - removal of a portion of the damaged strand, leaving single-stranded DNA
   - polymerization over the gap by DNAp, using the undamaged strand as a template
   - sealing of the DNA by DNA ligase, using ATP as the source of energy
- specialized enzymes
   - apurinic (AP) endonuclease: recognizes, excises depurinated nucleotide
   - uracil-DNA glycosylase: recognizes, hydrolyzes uracil base, allowing AP endonuclease to excise abasic site
DNA repair defects and cancer
- inherited defects in repair pathways increase susceptibility to cancer
   - exponential growth: a single defect increases likelihood of next defect, which increases likelihood of next defect
   - typically, defects in 5-6 DNA repair pathway genes are required before cancers begin to appear
- oncogenes: genes that promote unregulated cell growth (accelerate growth)
   - proto-oncogenes: promote regulated growth of normal cells
   - dominant mutations: only one defective allele is required for manifestation of disease
   - analogy: cellular gas pedal
- tumor suppressor genes: genes that allow unregulated cell growth only when deactivated (repress growth)
   - normal function: repress cell proliferation
   - recessive mutations: both alleles must be deactivated for manifestation of disease
   - analogy: cellular brake pedal
nucleotide excision repair: xeroderma pigmentosum
- xeroderma pigmentosum
   - cause: defect in nucleotide excision repair pathway
   - symptoms: hypersensitivity to sunlight, 2000-fold increase in skin cancer frequency
- nucleotide excision repair (NER) pathway
   - process
      - specialized proteins identify, bind the particular lesion
      - DNA endonucleases, directed by bound proteins, create single strand breaks 5’ and 3’ of lesion
      - helicase displaces lesioned oligodeoxynucleotide
      - gap is paired as in the common repair pathway
   - this process acts on a number of different DNA damages
- specific defects in XP
   - XP-A through XP-G
      - A, C: pyrimidine dimer recognition
      - F, G: DNA endonucleases (5’, 3’ respectively)
      - B, D: DNA helicases
   - XP-V gene: specialized DNAp error-free bypass that bypasses thymine dimers with AMP residues
      - with homozygous defect in this gene, only error-prone bypass or cell death can occur
      - as skin absorbs most UV radiation, that is most susceptible to disease (cell death, cancer)
mismatch repair: colorectal cancer
- colorectal cancer: cancer of the epithelium lining the colon and rectum
   - accounts for 10% of all cancer defects in the US
   - contain mutations in several of the known proto-oncogenes and tumor supressor genes
   - most are spontaneous, though 1 in 8 are hereditary non-polyposis colorectal cancer (HNPCC)
- inheritance of HNPCC: defect in MSH2
   - mutation: autosomal recessive inheritance
   - predisposition: autosomal dominant inheritance
      - having one defective allele (inherited dominantly) increases predisposition to disease (manifests recessively)
- MSH2: mismatched base pair repair sequence
   - substrate: single base mismatches and/or small mismatched insertions that form single-stranded loops
   - directs assembly of protein complex that contains a single stranded endonuclease
   - this endonuclease recognizes the newly-synthesized strand, which is the more likely defective strand
   - exonuclease excises defective DNA, and repair proceeds as in the common pathway
      - note that this is an exonuclease rather than a helicase, as found in NER
      - this may happen because the DNA to be removed is not chemically altered
                                                                      Biochemistry: NOTES & OBJECTIVES (page 39 of 165)




22. Nucleotide Metabolism and its Control with Drugs
Study Guide
do the following:
- phosphate attachments
   - ribose 5-P: attached to 5C
   - PRPP: P attached to 5C, PP attached to 1C on bottom
- AMP catabolism to uric acid
   - schematic pathway (blood cells)
      - AMP  adenosine (H2O  Pi; nucleotide phosphatase)
      - adenosine  inosine (I) (H2O  NH3; adenosine deaminase)
      - inosine  hypoxanthine (Pi  ribose 1-phosphate; nucleoside phosphorylase)
      - hypoxanthine  xanthine (xanthine oxidase)
      - xanthine  uric acid (xanthine oxidase
   - base salvage pathway: utilizes hypoxanthine for purine synthesis
   - summary: adenine vs. guanine
      - adenine: AMP  A  I  hypoxanthine  xanthine  uric acid
      - guanine: GMP  G   guanine  xanthine  uric acid
you should know:
- inosine monophosphate (IMP): precursor to both AMP, GMP
- ribonucleotide reductase: catalyzes ribonucleotides to deoxyribonucleotides, with NDPs as substrates
- thymidilate synthase: catalyzes dUMP  dTMP
you should understand:
- gout
   - caused by an excess of uric acid which crystallizes in the joints rather painfully
   - allopurinol
      - competitive inhibitor of xanthine oxidase, resulting in more soluble xanthine or hypoxanthine
      - xanthine and hypoxanthine can simply be excreted
- 5-fluoro-dUMP
   - incorporation pathway
      - 5-fluorouracil  5-f-UMP (PRPP  PPi, pyrimidine PRT)
      - 5-f-UMP  5-f-UDP (ATP  ADP, nucleotide kinase)
      - 5-f-UDP  5-f-dUDP (XH2  X + H2O, ribonucleotide reductase)
      - 5-f-dUDP  5-f-dUMP (H2O  Pi, nucleotide phosphatase)
   - mechanism
      - covalently binds thymidilate synthase, halting conversion of dUMP  dTMP
      - this halts cell growth and slows growth of the tumor


Notes: Lecture and Reading
overview of nucleotide metabolism
- functions of nucleotides
   - genetic information:                   DNA, RNA
   - energy metabolism:                     ATP
   - intracellular signals:                 cAMP, cGMP, GTP
   - coenzymes:                             adenosine derivatives
   - activating metabolic intermediates:    aminoacyl-AMP
- nucleotide metabolism
   - amino acids  ribonucleotides
      - ribonucleotides  RNA (NTP, RNAp)  ribonucleotides (NMPs, nuclease, kinase)
      - ribonucleotides  deoxyribonucleotides
                                                                         Biochemistry: NOTES & OBJECTIVES (page 40 of 165)



         - deoxyribonucleotides  DNA  deoxyribonucleotides
         - deoxyribonucleotides  purine, pyrimidine bases
            - purine, pyrimidine bases  ribonucleotides (salvage pathway)
            - purine, pyrimidine bases  waste products
      - ribonucleotides  purine, pyrimidine bases  waste products
- nucleotide synthesis
   - de novo: from scratch, using amino acids and other small molecules (highly expensive)
   - base salvage: from body or diet; more efficient (relatively inexpensive)
recycling of nucleosides
- mechanism
   - NTPs  mRNA (PPi; RNA polymerase)
   - mRNA  NMPs (H2O, nuclease)
   - NMPs  NDPs (ATP  ADP, kinase)
   - NDPs  NTPs (ATP  ADP, kinase)
- enzymes
   - nucleotide kinase: add phosphate groups to NMPs and NDPs, often using ATP as a Pi donor
   - nucleoside kinase: converts ribonucleosides to ribonucleotides as NMPs
catabolism (degradation) of nucleotides
- excess nucleotides: will be degraded into ribose and nucleotide bases, and bases will be excreted
- nucleotide degradation: NMP to hypoxanthine
   - pathway: AMP to hypoxanthine (blood)
      - AMP  adenosine, or A (H2O, Pi; phosphatase)
      - adenosine  inosine, or I (H2O, NH3; adenosine deaminase)
      - inosine  hypoxanthine (Pi, ribose 1-P; nucleoside phosphorylase)
   - pathway: AMP to hypoxanthine (other tissues, such as muscle)
      - AMP  adenosine, or A (H2O, Pi; phosphatase)
      - inosine  hypoxanthine (Pi, ribose 1-P; nucleoside phosphorylase)
      - adenosine  inosine, or I (H2O, NH3; adenosine deaminase)
   - CMP, GMP, UMP degradation
      - similar pathways, but base is not modified
      - cytosine, guanine, and uracil are released
   - adenosine deaminase defect: severe combined immunodeficiency disease (SCID)
      - ADA defect decreases conversion of adenosine to inosine
      - this is particularly damaging to lymphocytes, though the reasons are not well understood
      - SCID: first disease with which gene therapy was attempted
- nucleotide degradation: hypoxanthine  uric acid
   - pathway
      - hypoxanthine  xanthine (xanthine oxidase)
      - xanthine  uric acid (xanthine oxidase)
   - uric acid
      - beneficial anti-oxidant maintained near its solubility limit in blood by active re-absorption from kidney
      - gout: caused by excess uric acid, which crystallizes painfully in the joints and kidneys
      - allopurinol: competitively inhibits xanthine oxidase; soluble xanthine and hypoxanthine are secreted
base salvage pathways
- base salvage pathway: overview
   - bases can freely cross cell membrane
      - in tissues where they are in excess, they can be degraded
      - they can then diffuse to a tissue where they are needed, and then be reincorporated into nucleotides
   - this is also how ingested bases, and pharmaceutical analogs, can be used as nucleotides
- base salvage pathway
   - ribose 5-phosphate: the backbone of base salvage and de novo nucleotide synthesis
   - enzymes in the base salvage pathway
      - activation: requires ribose 5-phosphate conversion to 5-phosphoribosyl-1-pyrophosphate (PRPP)
      - conversion to nucleotide: requires a phosphoribosyl transferase (PRT)
   - general pathway
      - ribose 5-phosphate  5-phosphoribosyl-1-pyrophosphate (ATP  AMP, Mg2+; PRPP synthetase)
                                                                         Biochemistry: NOTES & OBJECTIVES (page 41 of 165)



      - PRPP + nucleotide base  NMP (PPi; nucleotide PRT)
      - NMP  NDP  NTP (ATP  ADP, nucleotide kinase)
   - specific pathways: hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
      - guanine: guanine  GMP (PRPP  PPi; HGPRT)
      - inosine: hypoxanthine  IMP (PRPP  PPi; HGPRT)
         - hypoxanthine is intermediate in structure between adenine, guanine
         - it can therefore be used as a precursor to both AMP and GMP
- clinical problems: HGPRT deficiency
   - partial decrease: increased uric acid, susceptibility to gout
   - complete deficiency: Lesch-Nyhan syndrome
      - characterized by mental retardation, spastic cerebral palsy, and propensity for self mutilation
      - also leads to gout, usually death by kidney failure
   - X-linked recessive trait: essentially restricted to males
deoxyribonucleotide synthesis from ribonucleotides
- general pathway
   - NDP  dNDP (ribonucleotide reductase)
      - note: ribonucleotide reductases only work on NDPs
   - dNDP  dNTP (nucleotide kinase)
- specific pathway: formation of dTTP from dUDP
   - dUDP  dUMP (nucleotide phosphatase)
   - dUMP  dTMP (thymidilate synthase)
   - dTMP  dTTP (nucleotide kinase)
- keeping dUTP levels low
   - because DNA polymerase does not distinguish between dUTP, dTTP, high [dUTP] is toxic to the cell
   - dUTPase: expressed by cells to hydrolyze dUTP to dUMP, thereby promoting thymidilate conversion to dTMP
   - binding affinities for dUTP
      - DNAp: Km = 10 μm
      - dUTPase: Km = 1 μm
      - the higher dUTPase affinity for dUTP helps assure dUTP will be held too low for common use by DNAp
- 5-fluoro-dUMP: cancer drug
   - function: irreversibly inhibit thymidilate synthase, indirectly halting cell division and slowing tumor growth
   - pathway: base salvage
      - 5-flurouracil  5-F-UMP (PRPP  PPi; pyrimidine PRT)
      - 5-F-UMP  5-F-UDP (ATP  ADP; nucleotide kinase)
      - 5-F-UDP  5-F-dUDP (XH2  X + H2O; ribonucleotide reductase)
      - 5-F-dUDP  5-F-dUMP (H2O  Pi; nucleotide phosphatase)
   - 5-F-dUMP covalently binds thymidilate synthase during reaction with the enzyme, halting it




23. Retroviruses and Gene Therapy
Study Guide
do the following:
- infective cycle of a simple retrovirus
   - virus recognized, taken into cell, and reverse transcribed from single RNA to double DNA
   - double-stranded DNA provirus is integrated into host chromosome, where it is transcribed
   - protein-coding genes used to make encapsidation proteins, which encapsidate the genomic RNA
   - encapsidated virus particles exit cell, look for more targets
- SCID and ADA: first trial
   - surviving T cells removed from patient, infected with retroviruses containing functional ADA genes
   - recombinant T cells infused into patient, giving a marked improvement in clinical condition and T cell count
   - improvements: infect progenitor cells, rather than differentiated T cells, to reduce the need for repeated infusions
- HSV thymidine kinase and ganclovir
                                                                       Biochemistry: NOTES & OBJECTIVES (page 42 of 165)



  - ganciclovir cannot be recognized by cellular nucleoside kinases in order to be activated
  - HSV tk does recognize it, however, converting it to a dGMP analog which cellular kinases will recognize
  - by using gene therapy, the HSV tk gene can be incorporated directly into tumor cells
  - with infusion of ganciclovir, tumor cells take substance in, where it works to halt DNA replication

you should know:
- creation of a vector
   - recombinant DNA technology is used to make a viral plasmid containing encapsidation signal, foreign gene
   - helper cells are created with helper plasmids containing encapsidation proteins, defective encapsidation signal
   - helper cells are infected with vector plasmids
   - helper plasmid proteins encapsidate vector genomic DNA, creating mature virus particles that can be collected
- advantages and disadvantages of retroviruses
   - small genome: easily manipulated, but can’t accept large genes
   - infects wide range of tissues: greater target possibilities, but more difficult to be specific
   - incorporation into host DNA: more stable expression of foreign gene, but can also modify normal expression
   - requires cell division: easier to target cancers, but harder to work in differentiated tissues


Notes: Lecture and Reading
gene therapy: general considerations
- use
   - gene therapy: introduction of genes into a patient as a means of treating or preventing a disease
   - targets: somatic cells
      - hereditary mutations would still be passed on to progeny
      - genetic modification of germ cells in humans is currently non-viable
- technique: insert the gene into a viral genome and use it to infect target cells
   - vector: virus that has been engineered for the purpose of infecting cells with specific genes
   - retroviruses
      - contain an RNA genome that is copied into DNA (reverse transcriptase) after entering a cell
      - commonly used vector in gene therapy
   - DNA cloning: used in gene therapy to isolate large amounts of a gene, insert it into an appropriate vector
infective cycle of the retrovirus and its inhibition by AZT
- characterization based on regulatory mechanisms
   - simple: Moloney Murine Leukemia Virus (MMLV); widely understood, commonly used
   - complex: Human Immunodeficiency Virus (HIV); regulation poorly understood, but could be useful in the future
- infective cycle of the retrovirus
   - entrance: virus uses cell surface receptors to gain entrance into the cell
   - reverse transcription: reverse transcriptase; single-stranded RNA genome  double-stranded DNA (provirus)
   - integration: provirus enters cell nucleus, recombines into host chromosome to become part of host DNA
   - transcription: cellular RNAp II generates viral genomic RNA and viral pre-mRNA
   - splicing: viral pre-mRNA is used to generate mature mRNA
   - translation: viral proteins are made from mRNA
   - encapsidation: viral proteins encapsulate viral genome, forming virus particles (RNA core, protein capsule)
   - budding: virus particles bud from cell surface, becoming surrounded in membrane envelope
- HIV retrovirus: inhibition by AZT
   - ddNTP: dideoxy nucleotide triphosphates that, when incorporated into DNA, halt synthesis
   - HIV reverse transcriptase, a sloppy enzyme, has an unusually high affinity (low K m) for certain ddNTPs
      - AZT: 3’-azido-2’,3’-dideoxythymidine
      - DDI: 2’,3’-dideoxyinosine
   - cellular DNAp has a much higher Km for AZT, DDI, and is thus less sensitive to these drugs
retroviral vectors and their use in gene transfer
- simple retrovirus genome
   - three protein
      - gag: capsid protein
      - pol: reverse transcriptase
      - env: membrane envelope protein
                                                                        Biochemistry: NOTES & OBJECTIVES (page 43 of 165)



   - cis-acting elements
      - used in reverse transcription, integration, proviral transcription, RNA processing, translation, encapsidation
      - typically found in long terminal repeats (LTRs) near the end of the genome
- construction of a vector provirus
   - elements
      - DNA cloning: process by which large quantities of a specific DNA fragment can be made in bacterial cells
      - plasmids: small, circular, extragenomic DNA naturally found in bacteria
      - restriction enzymes: used to cut DNA at specific sites
      - DNA ligase: used to ligate digested DNA
   - process
      - plasmid is digested at specific restriction enzyme sites
      - proviral DNA is inserted into the plasmid, and DNA ligase is used to ligate the fragments
      - therapeutic gene is inserted in place of the viral genes in further steps of digestion, ligation
      - recombinant plasmid is introduced into bacterial cells, and cells are cultured
- construction of an encapsidation-defective helper provirus
   - vector provirus lacks the proteins for encapsulation, though it contains the receptors to be encapsulated
   - encapsulation signal: cis-acting genomic element by which viral genome is recognized
   - in helper cells, the original provirus has its encapsulation signal knocked out
   - thus the helper provirus can create the packaging proteins, but cannot itself be packaged
- construction of a vector
   - proviral DNA form of genome is inserted into a bacterial plasmid
   - the encapsidation proteins are removed, a foreign gene is inserted, and the DNA is ligated
   - helper cells are made by inserting the E- helper provirus plasmid into bacteria
   - vector plasmid is inserted into helper cells
   - E- helper provirus directs expression of encapsidation proteins, which encapsidate vector genome to make virus
   - virus particles are collected from cell culture, used for gene transfer
- thus, for the sake of avoiding out of control infection, the vector virus is initially split
   - vector genome: contains foreign gene, encapsidation signal, and can only be used for genetic transfer
   - helper provirus: contains encapsidation genes, no encapsidation signal, can only be used for initial encapsidation
- advantages and disadvantages of retroviral vectors
   - small genome: easily manipulated, but cannot accept large genes
   - infects broad range of tissues: can be used for many diseases, but is harder to target with specificity
   - host integration of proviral DNA: stable maintenance of therapeutic gene, but can alter cellular gene expression
   - integration requires cell division: helps to target cancer cells, but limits use in differentiated tissues
gene transfer trials
- first trial: severe combined immunodeficiency disease (SCID) due to inherited adenosine deaminase (ADA) defect
   - process
      - surviving T lymphocytes were isolated
      - retroviral vector was used to transfer a functional ADA gene into T cells
      - T cells were reintroduced to bloodstream
   - results
      - gave a marked improvement in clinical condition, T cell count
      - however, required repeated infusions, as T cells typically survive 3-6 months
      - desire: incorporate ADA into progenitor cells, so that repeated infusions would not be necessary
- subsequent trials: SCID due to X-linked absence of functional gamma chain of CD3
   - results: 9 of 11 boys successfully treated
   - complications: 2 of these developed T cell leukemia 30 – 34 months after therapy
      - in one cell, gene had incorporated near LMO2, a proto-oncogene involved in hematopoiesis
      - this acted as a transcription activator, leading the cell to grow more frequently and be selected for
      - through normal mechanisms of cancer, it picked up other mutations and became cancerous
- cancer treatments: ganciclovir
   - function: halts DNA replication when incorporated into replicating DNA
   - problem
      - active form of ganciclovir (dGTP analog) cannot be taken in by cells
      - however, mammalian nucleoside kinases do not recognize inactive form, cannot activate it
   - solution: herpes simplex virus (HSV) thymidine kinase (tk)
                                                                        Biochemistry: NOTES & OBJECTIVES (page 44 of 165)



     - HSVtk has a broad substrate specificity, and can convert ganciclovir to a dGMP analog
     - cellular nucleotide kinases can then recognize, convert to dGTP analog, where it can be used
  - trial use in gene therapy
     - brain tumors were directly injected with a retrovirus containing an HSV tk gene
     - after integration into tumor DNA, ganciclovir was administered
     - only tumor cells expressing HSV tk used ganciclovir
  - results: now in phase III trials with 200 patients enrolled




25. Membrane Structure and Transport
Study Guide
do the following:
- phosphoglyceride structure
   - glycerol 3-phosphate
   - head group: polar alcohol, phosphodiester linkage
   - fatty chains: fatty acids, ester linkage
- major classes of lipids and membrane proteins
   - phosphoglycerides: glycerol 3-phosphate linked to two fatty acids and a polar alcohol group
   - sphingolipids: sphingosine ester-linked to polar head group, amide-linked to fatty acid
      - sphingomyelin: phospocholine head group
      - glycosphingolipids: sugar residues in head groups; often involved in cell-cell signaling
         - cerebrosides: glucose, galactose as a head group
         - gangliosides: complex oligosaccharide head groups
   - cholesterols: small polar hydroxyl head group, nonpolar steroid rings, nonpolar hydrocarbon tail
understand the following:
- membranes
   - membrane structure: aliphatic lipids forming a bilayer, with hydrophobic tails congregating at the center
   - membrane fluidity: determined by lipid composition, with increased saturation leading to decreased fluidity
   - membrane asymmetry: lipids and proteins are unable to spontaneously flip within the bilayer
- transport proteins
   - channels:       gated pores with access regulated by specific signals          diffusion-limited
   - transporters: physical binding and facilitated transport                       protein kinetics
- transport
   - passive: solute down an electrochemical gradient
   - active: transport against an electrochemical gradient, requiring coupling to energy source


Notes: Lecture and Reading
the composition of membranes
- composition
   - primary component: lipids, proteins
   - secondary component: carbohydrate (usually as glycolipid or glycoprotein)
- fluid mosaic model: lipid bilayer
   - function: barrier to most macromolecules, as well as common nutrients (glucose, amino acids)
   - lipids: bilayer structure of membrane, as well as fluid nature
   - proteins: diffuse in two dimensions, incapable of passive directionality changes, responsible for transport
lipid components: phosphoglycerides, sphingolipids, and cholesterol
- lipid components: overview
   - amphipathic: molecules containing both polar (hydrophilic) and nonpolar (hydrophobic) regions
   - polar lipids: amphipathic lipids such as those in biological membranes
   - in aqueous solutions, hydrophobic tails oriented away from water, with polar ends interacting with water
   - three classes: phosphoglycerides, sphingolipids, cholesterol
                                                                         Biochemistry: NOTES & OBJECTIVES (page 45 of 165)



- phosphoglycerides
   - general structure: glycerol 3-phosphate (G3P) linked to two fatty acids (R1, R2) and a polar alcohol group (X)
      - fatty acids:            ester linkage              –O–C(=O)–R
      - polar alcohol group: phosphodiester linkage        –O–P(=O, –OH)–O–X
   - fatty acyl groups
      - fatty acid composition
         - R1: usually saturated
         - R2: usually unsaturated
         - examples
            - palmitic:         C16
            - stearic:          C18
            - oleic:            C18Δ9
      - chain length
         - non-polarity: longer chain length increases
         - melting point: increases with increasing non-polarity (chain length)
         - membrane fluidity: influenced by melting point of lipids within the lipid bilayer
      - degree of unsaturation
         - Δn: double bond at position n to (n+1)
         - melting point: decreases with increasing degree of unsaturation
            - animal fats: high melting point (numerous saturated acids)
            - plant fats: lower melting point (unsaturated fatty acids)
      - common fatty acids in human phosphoglycerides
              common name           saturation                    carbons       abbreviation     melting point (°C)
              palmitic              saturated                       16              C16                  63
              stearic               saturated                       18              C18                  70
              oleic                 monounsaturated                 18             C18Δ9                 13
              linoleic              polyunsaturated                 18            C18Δ9,12                5
                                               9    9,12
      - memorization: PSOL (16, 18, 18 Δ , 18Δ )

   - polar alcohol groups
      - choline
      - ethanolamine
      - serine
      - glycerol
      - inositol
- sphingolipids
   - localization: found in all tissues, especially abundant in neural tissue (defects in metabolism give retardation)
   - general structure: sphingosine ester-linked to polar head group, amide-linked to fatty acid
      - sphingosine: long structure with a non-polar tail
      - overall appearance: similar to phospholipid, but without glycerol, and only one varying tail
   - sphingomyelin: phosphocholine head group
      - phosphocholine ester-linked to terminal alcohol moiety of sphingosine
      - structure, properties similar to phosphatidylcholine
   - glycosphingolipids: one or more sugar residues in head group
      - cerebrosides: glucose or galactose as a head group
      - gangliosides: large, polar head groups composed of complex oligosaccharides
         - involved in recognition events at cell surface
         - ABO blood type specified bye glycosphingolipids, other glycoproteins present on RBC surface
- cholesterol
   - general structure: small polar hydroxyl head group, nonpolar steroid rings, nonpolar hydrocarbon tail
   - steroid rings: rigid, increase membrane rigidity
effects of lipid composition on membrane structure and fluidity
- asymmetry of lipids in bilayer
   - lateral movement: free within plane of bilayer
   - vertical movement: lipids do not flip flop from one side to another
                                                                     Biochemistry: NOTES & OBJECTIVES (page 46 of 165)



   - this gives membranes asymmetrical surface based on synthesis
- membrane fluidity
   - two states: liquid, gelatin
   - biological membranes, physiological temperature: fluid (like olive oil)
   - cis double bonds: increase fluidity (kink in packing)
   - cholesterol: decrease fluidity (tighter packing between C1-C10)
- fluidity and function
   - membranes must be fluid so that proteins may collide in multi-step processes
   - some proteins are anchored to components of cytoskeleton
   - this imparts a differentiation of the plasma membrane, as seen in polar cells
integral and peripheral membrane proteins
- peripheral
   - general structure: similar to cytoplasmic proteins, with polar surface side chains
   - held to membranes through electrostatic, other weak interactions with lipids or integral membrane proteins
- integral (IMPs)
   - general structure: amphipatic polypeptides with one or multiple hydrophobic portions embedded in membrane
      - hydrophobic regions often composed of α helices traversing the bilayer
      - most IMPs extend all the way through the bilayer
   - single transmembrane domain
      - example: β subunit of insulin receptor
      - other examples: epidermal growth factor (EGF) receptor, low density lipoprotein (LDL) receptor
      - function: often bind extracellular signal molecule, transmit response to cellular interior
   - multiple transmembrane domains
      - G protein receptors (guanine nucleotide binding protein): seven transmembrane helices
         - visual rhodopsin
         - β adrenergic receptor
      - transport proteins: cystic fibrosis transmembrane conductance regulator (CFTR)
membrane transport
- transport
   - lipid bilayer: impermeable barrier for most molecules, allowing regulation of entry and exit
   - transport proteins: selectively take in useful components, dispose of less useful components
- rate of membrane transport
   - permeability
      - permeable:             gases, small molecules (O2, CO2)
      - semi-permeable:        H2O (though aquaporins also facilitate this)
      - impermeable:           large charged, uncharged molecules
   - transporters vs. channels
      - channels: gated pores with access regulated by specific signals (e.g. CFTR Cl - channel)
      - transporters: facilitated transport by binding on one side of membrane, physical transport to other side
         - saturation kinetics similar to enzymes
- energetics of transport
   - transport proteins cannot alter thermodynamics, only the rate
                                                                       [X B ]
      - chemical concentration gradient:                   ΔG  RTln
                                                                       [X A ]

    - membrane electrical potential:                 ΔG  ZF
      - Z: charge/mol
      - F: Faraday constant (23 kcal/volt·mol)
      - Ψ: difference in charge across membrane

                                                                 [X B ]
    - total electrochemical potential:               ΔG  RTln           ZF 
                                                                 [X A ]
  - use
     - chemical:           favorable (lower ΔG) from high to low concentration
     - electrochemical:    favorable when moving towards electrochemical neutrality
                                                                       Biochemistry: NOTES & OBJECTIVES (page 47 of 165)




26. Membrane Transport Systems
Study Guide
understand the following:
- glucose carriers
   - SGLT-1:         active glucose-Na+ cotransporter               intestine
   - GLUT-1:         passive basal uptake                           RBC, brain
   - GLUT-2:         passive homeostasis                            intestine, liver, kidney, pancreas (β cells)
   - GLUT-3:         passive                                        brain, kidney, placenta
   - GLUT-4:         passive insulin-mediated                       muscle, fat
- Na+/K+-ATPase
   - normal concentrations: high intracellular K+, extracellular Na+
   - per ATP: 3 Na+ out, 2 K+ in
- intestinal, kidney epithelial transport
   - Na+/K+-ATPase:           creates a Na+ electrochemical gradient by driving Na+ out of cell into blood
   - SGLT-1:                  actively carries glucose into epithelial cells in symport with Na +
   - GLUT-2:                  passively carries glucose from epithelia into bloodstream (homeostatic transporter)
- cystic fibrosis transmembrane regulator (CFTR)
   - function: passive transport of Cl- at apical membranes of epithelial cells
   - airway obstruction: thick mucus
      - normal
         - NaCl transporter: brings Na+, Cl- ions into cell from basal membrane
         - Na+ channel:       secretes Na+ into lumen
         - CFTR:              secretes Cl- into lumen
         - aquaporins:        water follows into lumen, decreasing mucus viscosity
      - cystic fibrosis
         - CFTR:              BLOCKED
         - Na+ channel:       absorbs Na+ from lumen
         - NaCl transporter: UNFAVORABLE; does not transport Na+, Cl- ions
         - aquaporins:        water follows into cell, increasing mucus viscosity
   - sweat glands: high Cl-
      - normal
         - secretory coil:    Na+/Cl- transporter, some cells have CFTR, some cells have Ca2+-activated transporters
         - reabsorptive duct: Na+/K+-ATPase creates gradient to draw in sodium, Cl- follows through CFTR
      - cystic fibrosis
         - secretory coil:    CFTR blocked, but Ca2+-activated transporters still release Na+
         - reabsorptive duct: Na+/K+-ATPase creates gradient to bring in sodium, but CFTR cannot bring in Cl -


Notes: Lecture and Reading
glucose transport uses passive and active transport systems
- transport against electrochemical gradients
   - active transport: makes electrochemically unfavorable transport possible by linking to metabolic energy
      - ATP hydrolysis: used by Na+/K+ transporters
      - ion gradient: used by glucose transporters in kidney, driven by Na + electrochemical gradient
   - three types of transporters mediate glucose intake
      - SGLT: active transport of glucose in epithelial cells coupled to favorable Na + gradient
      - Na+/K+-ATPase: use of ATP hydrolysis to maintain the Na + and K+ gradients
      - GLUT: passive transport of glucose
- GLUT family of glucose transporters: mediates passive transport of glucose in many tissues
   - GLUT-1: glucose transporters of RBCs
                                                                         Biochemistry: NOTES & OBJECTIVES (page 48 of 165)



     - structure: 12 transmembrane helices, typical of GLUT family
     - function: increase rate of transport by binding on one side, releasing to other side
     - kinetics: saturation kinetics typical of an enzyme
        - Km: 2 mM
        - post-meal level of D-glucose: 5 mM
        - because transporter facilitates equilibration, high blood glucose can be passively transported in
        glucoseOUT + GLUT-1 ↔ [glucose-GLUT-1] ↔ glucoseIN + GLUT-1
  - glucose transporters in other tissues
     - overview: important points
        - brain:             GLUT-3 and GLUT-1
        - muscle, adipose: GLUT-4, slightly higher than RBC carrier
        - insulin:           increases number of GLUT-4 present in plasma membrane of muscle, adipose tissue
        - liver:             GLUT-2, high Km allows metabolism at high portal glucose concentration
     - summary
             transporter type                       tissue                            Km (mM)       function
             SGLT-1        active: Na+ cotrans.     small intestine, renal tubules    0.1-10        active transport
             GLUT-1        passive                  ubiquitous, esp. RBC and brain 1-2              basal cellular uptake
             GLUT-2        passive                  liver, small intestine, kidney,   15-20         glucose homeostasis
                                                    pancreatic β cells
             GLUT-3        passive                  many tissues, esp. brain,         <1
                                                    placenta, kidney
             GLUT-4        passive                  muscle, adipose                   ~5            insulin-mediated
                                                                                                    glucose uptake

- SGLT family of glucose transporters Na+ gradient coupled to glucose transport
   - function: promotes active uptake of glucose in kidney, small intestine
   - intestinal epithelial structure
      - brush border surface:           SGLT             Na+-glucose active transporter
      - serosal surface:                  +   +
                                        Na /K -ATPase establishes Na+ gradient
                                        GLUT-2           allows passive glucose transport following Na+ gradient
   - process
      - glucose brought into epithelial cells by sodium cotransport (SGLT)
      - sodium actively pumped into blood vessel lumen (Na +/K+-ATPase)
      - glucose follows passively (GLUT-2)
- Na+/K+-ATPase and ion transport
   - animal cell approximate ion concentrations
      - intracellular:         [K+] = 120-160 mM         [Na+] = 10 mM
      - extracellular:         [K+] = 5 mM               [Na+] = 150 mM
                                     +  +
   - maintaining the gradient: Na /K -ATPase
      - 3 Na+IN + 2 K+OUT ↔ 3 Na+OUT + 2 K+IN (Mg2+; ATPADP + Pi)
      - sodium pumped out, potassium pumped in
   - related energy usage
      - brain: 2% of body weight, 20% of O2 use of body
      - 2/3 of that 20% is simply to maintain the transmembrane ion concentrations
   - asymmetry of enzyme
      - Na+: binds on cytoplasmic surface
      - K+:     binds on extracellular surface
      - ATP: binds only on cytoplasmic surface
CFTR mediates passive transport of Cl-
- CFTR: overview
   - cystic fibrosis transmembrane regulator
   - function: passive transport of Cl- at apical membranes of epithelial cells
   - distribution: airway mucosa, pancreas, salicary glands, sweat glands
   - consequences of defects
      - mucus lining becomes more viscous
                                                                       Biochemistry: NOTES & OBJECTIVES (page 49 of 165)



      - pancreas: plugs up pancreatic duct, leading to a deficiency of pancreatic enzymes
      - lung: makes lungs more susceptible to infection
- CFTR: structure
   - MSD: 12 transmembrane segments organized into 2 membrane spanning domains (MSD 1, 2)
   - NBD: nucleotide binding domains, sites of binding nucleotides, required for Cl - transport
   - R: location of cyclic AMP-dependent phosphorylation as required for channel opening
- CFTR: mutations
   - class I: absence of synthesis
   - class II: defective maturation and degradation
   - class III: defective regulation
   - class IV: defective chloride conductance or channel gating
   - class V: decreased transcripts due to splicing defects
   - class VI: accelerated cell surface turnover
   - most common Caucasian mutation: ΔF508
CFTR in the airway epithelia: physiological consequences
- normal
   - Na+, Cl- taken in by cell from body
   - Cl- expelled, water follows, leading to a less viscous mucous
- abnormal
   - Cl- unable to be transported out, so intake from body of Na +, Cl- becomes unfavorable
   - Na+ taken in from apical surface instead, which brings in water
   - this leads to a more viscous extracellular mucus
CFTR in sweat glands epithelia: the sweat test
- process
   - to drive out Na+, Cl- is secreted in secretory coils of sweat ducts by CFTR channels, Ca2+-activated channels
   - excess Cl- is reabsorbed by CFTR cells in the reabsorptive duct
   - defects in CFTR lead to a deficiency in reabsorption, yielding higher [Cl -] in sweat
- diagnosis: use higher Cl- concentrations as a marker of the disease, given other symptoms




27. Glucose Metabolism
Study Guide
you should understand:
- catabolism
   - strategy
      - release energy from oxidation of fuel (nutrient) sources
      - conserve energy through formation of phosphoanhydride bond in ATP
      - energy release: oxidation of the substrate, conserved in a reduced acceptor (oxidizing agent)
   - common electron carriers
      - nicotinamide adenine dinucleotide (NAD+)
         - reactive portion: nicotinamide ring, accepts 1 H: (H atom plus 1 e-)
         - common use:       dehydrogenation of an alcohol to a ketone
         - origin:           niacin (deficiency causes pellagra)
      - flavin adenine dinucleotide (FAD)
         - reactive portion: nitrogens across -N=C-C=N- in ring structure, accepts 2 H· (hydrogen atoms)
         - common use:       dehydrogenation of an alkane to alkene
         - origin:           riboflavin (vitamin B2) (no major associated disease)
- energetics
   - reversibility
      - irreversible:        ΔG values of great magnitude
      - reversible:          ΔG values of relatively small magnitude (close to 0)
   - coupled reactions
                                                                     Biochemistry: NOTES & OBJECTIVES (page 50 of 165)



      - calculate ΔG of individual reactions, ensuring that signs are correct
      - sum values to get overall ΔG
you should know
- glycolysis
   - recognize glucose and the 10 intermediates
   - know the types of reactions
      - (1) hexokinase:                         phosphorylation (IRREVERSIBLE)
      - (2) phosphoglucose isomerase:           isomerization (aldose  ketose)
      - (3) phosphofructokinase:                phosphorylation (IRREVERSIBLE)
      - (4) aldolase:                           aldol cleavage of a C-C bond
      - (5) triose phosphate isomerase:         isomerization
      - (6) G 3-P dehydrogenase:                dehydrogenation, phosphorylation
      - (7) phosphoglycerate kinase:            ADP phosphorylation
      - (8) phosphoglycerate mutase:            isomerization (mutase)
      - (9) enolase:                            dehydration
      - (10) pyruvate kinase:                   ADP phosphorylation (IRREVERSIBLE)
   - draw structure of product or reactant, given the other
- irreversible reactions of glycolysis
   - hexokinase:             glucose  glucose 6-P
   - phosphofructokinase: fructose 6-P  fructose 1,6-bis-P
   - pyruvate kinase:        phosphoenolpyruvate  pyruvate


Notes: Lecture and Reading
introduction to metabolism
- overview
   - ATP: commonly used energy storage molecule
      - nutrients taken in, energy extracted to make ATP
      - energy: stored in the large free energy change associated with hydrolysis of phosphoanhydride bonds
   - metabolic pathways
      - anabolic: biosynthetic
         - reductive, require expenditure of free energy (usually as ATP)
         - common electron donor: nicotinamide adenine dinucleotide phosphate (NADPH)
      - catabolic: degrative
         - oxidative, use organic molecules to make ATP
         - oxidation: usually involved, as usable energy is derived from increasing oxidation state of carbon
- physiological importance of glucose metabolism
   - glucose: only source of energy for brain, RBCs
   - normal conditions: blood glucose at 65-110 mg / 100 mL blood (3.6-6.1 mM)
      - eating: diet is primary glucose source
      - non-eating: glucose derived from breakdown of glycogen stores, gluconeogenesis
- glucose as a fuel source
   - glucose is an excellent fuel source because:
      - (1) abundant in diet
      - (2) large predicted standard free energy change (ΔG’˚= -686 kcal/mol) for complete oxidation to CO2, H2O
   - subjected to glycolysis, pyruvate DH, citric acid cycle, electron transport, oxidative phosphorylation
   - free energy conserved as 36-38 ATP per glucose
oxidation-reduction reactions
- catabolic strategy
   - release energy from oxidation of fuel (nutrient) sources
   - electrons removed, transferred to an electron acceptor (oxidant) that itself becomes reduced
- nicotinamide adenine dinucleotide (NAD+)
   - reactive portion: nicotinamide ring
      - NAD+ + H:  NADH
      - H: = one hydrogen atom plus one electron
   - reactions using NAD+ as an electron acceptor
                                                                       Biochemistry: NOTES & OBJECTIVES (page 51 of 165)



      - dehydrogenation of an alcohol to a ketone
      - example: lactate  pyruvate (lactate dehydrogenase; NAD+  NADH + H+)
   - origin: niacin
      - niacin: essential vitamin found in unrefined and enriched grains, cereal, milk, lean meats
      - pellagra: deficiency of niacin affecting skin, GI tract, CNS
- flavin adenine dinucleotide (FAD)
   - reactive portion: nitrogens across -N=C-C=N- in ring structure
      - FAD  FADH2 (2 H· )
   - reactions using FAD as an electron acceptor
      - dehydrogenation of an alkane to alkene
      - example: succinate  fumarate (FAD  FADH2)
   - origin: riboflavin (vitamin B 2)
      - source: milk, eggs, liver, green leafy vegetables
      - deficiency of riboflavin not associated with a major human disease
GLUCOSE METABOLISM
structure of glucose
- formula: (CH2O)n
   - basic unit of any sugar: H-C-OH
   - oxidation: will remove protons, providing energy to synthesize ATP
- carbonyl group: =O
   - all sugars have one carbonyl atom
      - aldoses (like glucose): terminal carbon
      - ketoses: internal carbon
   - by convention, C1 is the carbonyl carbon of glucose
- naming
   - sugars are named for the number of carbon atoms
   - sugars in this course
      - hexoses (6C):         glucose, galactose, fructose
      - pentoses (5C):        ribose
      - trioses (3C):         glyceraldehyde, dihydroxyacetone
- asymmetry
   - sugars: named for the asymmetrical carbon furthest from the carbonyl carbon
      - D: OH on right
      - L: OH on left
   - isomers: same chemical formula, but differ in the placement of atoms (e.g. glucose and fructose)
   - epimers: sugars that differ only by a single asymmetric carbon (e.g. glucose and galactose)
glycolysis
- formula: glucose + 2 ADP + 2 Pi + 2 NAD+  2 pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O
- ATP production: 2 ADP + 2 Pi  2 ATP
   - 2 ATP used, 4 ATP synthesized, with a net product of 2 ATP
   - substrate level phosphorylation: ATP produced by direct Pi transfer from substrate
- oxidation: 2 NAD+  2 NADH + 2 H+
   - 4 H+ atoms removed during glycolysis
   - NADH must be reoxidized to NAD+ for continuous use in cycle
      - in presence of O2: oxidative phosphorylation (and pyruvate metabolism)
      - in absence of O2: pyruvate converted to lactate, regenerating NAD+
reactions of glycolysis
- (1) hexokinase
   - reaction:       glucose  glucose 6-phosphate [ATP  ADP]
   - enzyme:         hexokinase
   - mechanism: phosphorylation
   - energetics: essentially irreversible
   - regulation: inhibited by glucose 6-phosphate
- (2) phosphoglucose isomerase
   - reaction:       glucose 6-phosphate  fructose 6-phosphate
   - enzyme:         phosphoglucose isomerase
                                                                  Biochemistry: NOTES & OBJECTIVES (page 52 of 165)



   - mechanism: isomerization (aldose  ketose)
   - energetics: reversible
- (3) phosphofructokinase
   - reaction:      fructose 6-phosphate  fructose 1,6-bisphosphate [ATP  ADP]
   - enzyme:        phosphofructokinase
   - mechanism: phosphorylation
   - energetics: irreversible, and first committed step of glycolysis
   - regulation: activated by AMP, inhibited by ATP
- (4) aldolase
   - reaction:      fructose 1,6-bisphosphate  dihydroxyacetone phosphate + glyceraldehyde 3-phosphate
   - enzyme:        aldolase
   - mechanism: aldol cleavage of a C-C bond
   - energetics: reversible
- (5) triose phosphate isomerase
   - reaction:      dihydroxyacetone 3-phosphate  glyceraldehyde 3-phosphate
   - enzyme:        triose phosphate isomerase
   - mechanism: isomerization
   - energetics: reversible
- (6) glyceraldehyde 3-phosphate dehydrogenase
   - reaction:      glyceraldehyde 3-phosphate  1,3-bisphosphoglycerate [H-OPO3H2; NAD+  NADH + H+]
   - enzyme:        glyceraldehyde 3-phosphate dehydrogenase
   - mechanism: oxidative phosphorylation: dehydrogenation (oxidation) of G3P accompanied by phosphorylation
   - energetics: reversible under cellular conditions
- (7) phosphoglycerate kinase
   - reaction:      1,3-bisphosphoglycerate  3-phosphoglycerate [ADP  ATP]
   - enzyme:        phosphoglycerate kinase
   - mechanism: cleavage of the carboxyl phosphate to phosphorylate ADP
   - energetics: reversible
- (8) phosphoglycerate mutase
   - reaction:      3-phosphoglycerate  2-phosphoglycerate
   - enzyme:        phosphoglycerate mutase
   - mechanism: isomerization by a mutase
   - energetics: reversible
- (9) enolase
   - reaction:      2-phosphoglycerate  phosphoenolpyruvate [H2O]
   - enzyme:        enolase
   - mechanism: dehydration to redistribute energy of molecule
   - energetics: reversible
- (10) pyruvate kinase
   - reaction:      phosphoenolpyruvate  pyruvate [ADP  ATP]
   - enzyme:        pyruvate kinase
   - mechanism: cleavage of high energy enol phosphate group to form ATP by substrate level phosphorylation
   - energetics: irreversible
   - regulation: inhibited by ATP
general principles
- balance of glycolysis reactions
   - 2 net ATP formed
   - 2 NADH formed from 2 NAD+
      - only small levels of NAD+ are maintained within a cell, so it must be constantly regenerated
      - this may be done anaerobically (through lactate formation) or aerobically (through the TCA cycle)
- irreversible reactions: large intracellular free energy changes
   - irreversible reactions
      - hexokinase:                    glucose  glucose 6-phosphate
      - phosphofructokinase:           fructose 6-phosphate  fructose 1,6-bisphosphate
      - pyruvate kinase:               phosphoenolpyruvate  pyruvate
   - these enzymes are allosterically regulated, as might be predicted
                                                                       Biochemistry: NOTES & OBJECTIVES (page 53 of 165)



- substrate level phosphorylation: formation of ATP using P i from substrate (e.g. G3P  3-PG)
   - (A) formation of a high energy bond: glyceraldehyde 3-phosphate dehydrogenase
      - reaction: glyceraldehyde 3-phosphate  1,3-phosphoglycerate
      - mechanism: aldehyde carbon oxidized to carboxyllic acid, forming a high energy carboxyl phosphate
         - (1) energy released from redox reaction:                     ΔG’˚ = -10.5 kcal/mol
         - (2) energy conserved by formation of carboxyl phosphate:     ΔG’˚ = +12 kcal/mol
         - net energy:                                                  ΔG’˚ = +1.5 kcal/mol
   - (B) generation of ATP by SLP: phosphoglycerate kinase
      - reaction: 1,3-bisphosphoglycerate  3-phosphoglycerate
      - mechanism: high energy phosphate bond cleaved, coupled to formation of ATP
         - (3) energy of hydrolysis:                                    ΔG’˚ = -12 kcal/mol
         - (4) energy of ATP formation:                                 ΔG’˚ = +7.5 kcal/mol
         - net energy:                                                  ΔG’˚ = -4.5 kcal/mol
   - mass action drives process forward




28. Pyruvate Metabolism
Study Guide
you should understand:
- anaerobic glucose metabolism: lactate dehydrogenase
   - reaction:       pyruvate  lactate [NADH + H+  NAD+]
   - enzyme:         lactate dehydrogenase
   - mechanism: ketone hydrogenation
   - result:         regeneration of NAD+ via formation of lactate
- aerobic glucose metabolism: ETS, oxidative phosphorylation
   - electron transport system:        utilizes electrons from mitochondrial NADH to drive H+ out of matrix
   - oxidative phosphorylation:        utilizes H+ gradient to turn the ATPase proton pump
- pyruvate transport into mitochondria
   - outgoing H+ gradient makes mitochondrial matrix more alkaline (basic)
   - electroneutrality causes OH- ions to want to exit
   - mitochondrial pyruvate-OH- antiporter couples outgoing OH- to incoming pyruvate
- pyruvate dehydrogenase complex
   - E1 complex: pyruvate decarboxylase
      - step 1: decarboxylation of pyruvate, transfer of fragment to TPP (product: CO 2)
   - E2 complex: dihydroprolyl transacetylase
      - step 2: oxidation of fragment via reduction of lipoic acid disulfide bond
      - step 3: transfer of fragment to CoA-SH (product: acetyl-CoA)
   - E3 complex
      - step 4: oxidative regeneration of lipoic acid via FAD, generating FADH 2
      - step 5: oxidation of FADH2, regeneration of FAD via NAD+, generating NADH + H+
- oxidative decarboxylation of α-keto acids: cofactor roles
   - TPP:            decarboxylates and attaches to substrate
   - lipoic acid: accepts substrate from TPP, attaches it to CoA-SH
   - CoA-SH:         attaches to fragment, forming a leaving group useful for later reactions
   - FAD:            oxidizes and regenerates lipoic acid, becoming FADH2 in the process
   - NAD+:           oxidizes FADH2, regenerating FAD, becoming NADH + H + in the process
- thiamine deficiency
   - disease:        Beri Beri
   - distribution: countries where polished rice is the staple diet (thiamine is removed in shavings)
   - pathology: accumulation of pyruvate
   - symptoms: irreversible brain damage
- regulation of pyruvate dehydrogenase: kinase/phosphatase system
                                                                      Biochemistry: NOTES & OBJECTIVES (page 54 of 165)



  - kinase:       p-PDH (inactive form); activated by reaction products acetyl-CoA, NADH
  - phosphatase:  PDH (active form)


Notes: Lecture and Reading
fates of pyruvate
- sources
   - glycolysis: oxidation of glucose
   - catabolism of amino acids
- fates
   - complete oxidation to CO2, H2O: TCA cycle
      - mitochondrion: contains enzymes for TCA cycle, electron transport, and oxidative phosphorylation
      - O2: final electron acceptor during electron transport process
   - incomplete oxidation
      - cells must rely on glycolysis ATP
      - NAD+ must be regenerated by other means
pyruvate to lactate
- conversion to lactate occurs when:
   - cells lack mitochondria:            RBCs, cornea, lens, regions of retina)
   - cells have few mitochondria: kidney medulla, testis, leukocytes, white muscle fiber
   - cells become limited in O2:         exercising muscle
- consequence of limited oxidation potential
   - glycolytic reactions become primary ATP source
   - NAD+ is regenerated from NADH by donation of electrons to pyruvate, forming lactate
   - enzyme: lactate dehydrogenase
- lactate dehydrogenase
   - reaction: pyruvate  lactate [lactate dehydrogenase: NADH + H+  NAD+]
   - structure
      - tetramer of two varying subunits, H and M
      - possible structures: H4, H3M, H2M2, HM3, M4
   - consequences
      - differ in catalytic, physical, and immunological properties
      - structure is tissue specific, and electrophoretic properties can be used to identify damaged organs
   - subunit predominance
      - H subunit: heart muscle
      - M subunit: skeletal muscle, liver
oxidative fate of pyruvate
- free energy changes: aerobic vs. anaerobic
   - anaerobic
      - glucose  2 lactate (ΔG’° = -47 kcal/mol)
      - conservation: 2 ATP
   - aerobic
      - glucose + 6 O2  6 CO2 + 6 H2O (ΔG’° = -686 kcal/mol)
      - conservation: 36-38 mol ATP
      - process requires:
         - oxidative decarboxylation of pyruvate to acetyl CoA
         - reactions of the TCA cycle
         - electron transport system
         - ATP synthesis by oxidative phosphorylation
      - process localization: mitochondria
- transport of pyruvate into mitochondria
   - mitochondrial structure
      - outer membrane: impermeable to large macromolecules, permeable to small molecules (metabolites)
      - inter-membrane space: between outer membrane, inner membrane
      - inner membrane: impermeable to most metabolites; transport must occur to bring into matrix
      - matrix: interior of the mitochondrion
                                                                       Biochemistry: NOTES & OBJECTIVES (page 55 of 165)



   - transport of pyruvate in
      - mitochondrial matrix: more alkaline (basic) than cytoplasm
      - antiporter: pyruvate exchanged for OH-, which follows favorable gradient
oxidative decarboxylation of pyruvate into acetyl CoA
- components
   - reaction: pyruvate + HS-CoA + NAD+  acetyl CoA + NADH + H+ + CO2
   - mechanism: oxidative decarboxylation
   - enzyme: pyruvate dehydrogenase
      - multi-enzyme complex of three enzymes
      - each enzyme requires a different cofactor for proper function
   - cofactors
      - thiamine pyrophosphate (TPP):           decarboxylation              E1 subunit
      - lipoic acid:                            electron transfer            E2 subunit
      - FAD:                                    electron transfer            E3 subunit
- reactions
   - E1 (pyruvate decarboxylation): pyruvate decarboxylase
      - [1] pyruvate decarboxylation, attachment of hydroxyethyl fragment to cofactor (TPP)
      - product: CO2 given off
      - yeast: hydroxyethyl fragment removed to form acetaldehyde, eventually ethanol
   - E2 (generation of acetyl-CoA): dihydroprolyl transacetylase
      - [2] oxidation of pyruvate C2 to form a thioester bond (cofactor: lipoic acid)
         - 2C fragment is oxidized by attachment to lipoic acid (concurrent reduction of disulfide bond)
         - TPP is released
         - oxidation state: 2C fragment equivalent to acetic acid
         - lipoic acid: bound to ε-amino group of lysine residue on E2
      - [3] transfer of 2C unit to HS-CoA
         - product: acetyl-CoA
   - E3 (regeneration of oxidized lipoic acid)
      - [4] reduced lipoic acid + FAD  oxidized lipoic acid + FADH2
      - [5] FADH2 (enzyme bound) + NAD+  FAD (enzyme bound) + NADH + H+
   - general principle: redox reaction
      - substrate oxidation used to create a high energy thioester bond (acetyl-CoA)
      - some energy of oxidation conserved as NAD+ reduction (NADH)
- coenzymes and vitamin precursors
   - coenzyme A
      - precursor:            pantothenic acid
      - deficiency:           rare in humans
      - active group:         sulfhydryl group on cysteamine (β-mercaptoethylamine) carries, transfers acyl groups
   - lipoic acid:             not a vitamin (required in some protozoa, bacteria, but not in humans)
   - thiamine pyrophosphate (TPP)
      - precursor:            thiamine (vitamin B1), formed by pyrophosphate transfer from ATP
      - sources:              meats, eggs, green vegetables, cereals, grains, nuts
      - required in:          oxidative phosphorylation, pentose phosphate
      - deficiency:           impairment of carbohydrate metabolism
                              neurotoxic accumulation of pyruvate
                              frequent CNS difficulties (especially given the central role of glucose in the brain)
      - disease:              Beri Beri: countries where polished rice is a staple food
                              Wernicke’s disease: alcoholics, other people with poor diets
      - storage:              humans cannot store excess thiamine, but excess thiamine is harmless
defects in pyruvate dehydrogenase (PDH)
- genetic defects
   - symptoms: similar to thiamine deficiency, with first effects seen in CNS
   - pathology: related to pyruvate, lactate accumulation in blood, with severity related to extent
      - most severe: fatal
      - more mild: developmental delays, mental retardation
- primary biliary cirrhosis
                                                                      Biochemistry: NOTES & OBJECTIVES (page 56 of 165)



   - pathology: antibodies created against pyruvate dehydrogenase E2 subunit
   - distribution: found primarily in women
regulation
- pyruvate DH subunits and function
   - E1:             step 1 (pyruvate decarboxylation, attachment to TPP)
   - E2:             step 2, 3 (generation of acetyl-CoA)
   - E3:             step 4, 5 (regeneration of lipoic acid)
   - kinase:         formation of inactive p-PDH
   - phosphatase: formation of active PDH
- regulation
   - kinase
      - mechanism:             phosphorylates Ser residue on E1
      - result:                inactivation of enzyme, reduction in acetyl-CoA and NADH production
      - regulation:            inhibited by acetyl-CoA and NADH
   - phosphatase
      - mechanism:             dephosphorylates Ser residue on E1
      - result:                activation of enzyme, increase in acetyl-CoA and NADH production
oxidative decarboxylations of other α-keto carboxylic acids
- general reaction: RCOCO2H + CoA-SH + NAD+  RCOS-CoA + NADH + H+ + CO2
- cofactors: TPP, lipoic acid, FAD
- examples
   - α-ketoglutarate DH (TCA cycle)
   - oxidative decarboxylation of α-ketobutyrate (amino acid catabolism)
   - oxidative decarboxylation of branched chain α-keto acids (formed from valine, leucine, isoleucine)




29. Tricarboxylic Acid (TCA) Cycle
Study Guide
you should know:
- TCA cycle
   - know the eight reactions of the TCA cycle (enzyme names, reaction types)
   - recognize structures of the eight cycle intermediates
- memorizing the TCA cycle
   - shorthand representation: P’ACwAwI’ α’SgSf FwMO
   - meanings
      - LETTER: denotes both the name of a cycle intermediate and an enzyme name
      - LETTER: denotes only the name of an enzyme
      - underline: enzymatic reactions in which NADH + H+ is made (PIαM)
      - (’):       enzymatic reactions in which CO2 is given off (PIα)
      - w:         enzymatic reactions in which water is a reactant (CAW)
      - g:         enzymatic reaction in which GTP is initially produced
      - f:         enzymatic reaction that uses FAD as an oxidant
you should understand:
- TCA cycle: acetyl-CoA to CO2
   - acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O  2 CO2 + HS-CoA + 3NADH + 3 H+ + FADH2 + GTP
   - component reactions:
      - acetyl-CoA  HS-CoA + 2 CO2
      - 3 NAD+  3 NADH + 3 H+
      - FAD  FADH2
      - GDP + Pi  GTP
      - 2 H2O
- TCA cycle and pyruvate metabolism: pyruvate to CO2
                                                                     Biochemistry: NOTES & OBJECTIVES (page 57 of 165)



   - pyruvate + 4 NAD+ + FAD + GDP + Pi + 2 H2O  3 CO2 + 4 NADH + 4 H+ + FADH2 + GTP
   - components:
      - pyruvate  3 CO2
      - 4 NAD+  4 NADH + 4 H+
      - FAD  FADH2
      - GDP + Pi  GTP
      - 2 H2O
- uses of the TCA cycle
   - acetyl-CoA: terminal oxidative pathway for CHO, fat, and protein metabolism
   - portions of the pathway used in numerous other metabolic conversions
- catalytic: oxaloacetate enters with acetyl-CoA, ultimately regenerating oxaloacetate
- oxidations: typically, 2 electrons removed as 2 H: (H+ + e-)
   - 3 NADH:        oxidative carboxylation of an α-keto acid (1)
                    alcohol to ketone dehydrogenation (2)
   - 1 FADH2:       alkane-alkene dehydrogenation
- substrate level phosphorylation: succinyl-CoA synthetase to give GTP (which isoenergetically gives ATP)
- regulation
   - isocitrate DH:          inhibited by NADH
   - citrate synthase:       inhibited by NADH, citrate


Notes: Lecture and Reading
importance of the tricarboxylic acid cycle
- alternate names: TCA cycle, citric acid cycle, Krebs cycle
- importance: focal point of all degradative pathways occurring in the cell
   - acetyl-CoA: end product of CHO, fat, and protein metabolism
      - most carbon atoms in our food eventually excreted as CO2
      - 2/3 of these: converted to CO2 in TCA cycle after conversion to acetyl-CoA
   - 2/3 of ATP used in body generated via TCA, electron transport, and oxidative phosphorylation
reactions of the citric acid cycle
- (1) citrate synthase
   - reaction:       acetyl-CoA + oxaloacetate  citrate [H2O ;  CoA-SH]
   - enzyme:         citrate synthase
   - mechanism: thioester hydrolysis
   - energetics: irreversible (first committed step)
   - result:         large drop in free energy
- (2) aconitase
   - reaction:       citrate  isocitrate [H2O ;  H2O]
   - enzyme:         aconitase
   - mechanism: sequential dehydrogenation/hydrogenation reaction; enzyme-bound cis-aconitase intermediate
   - energetics: reversible, with equilibrium citrate/isocitrate =13
   - result:         OH placed on carbon that is more easily oxidized in next reaction
- (3) isocitrate dehydrogenase
   - reaction:       isocitrate  α-ketoglutarate [ CO2; NAD+  NADH + H+]
   - enzyme:         isocitrate dehydrogenase
   - mechanism: oxidative (NAD+, alcohol to ketone) decarboxylation
   - energetics: irreversible
- (4) α-ketoglutarate dehydrogenase
   - reaction:       α-ketoglutarate + HS-CoA  succinyl-CoA [CO2; NAD+  NADH + H+]
   - enzyme:         α-ketoglutarate dehydrogenase (cofactors: TPP, lipoic acid, FAD)
   - mechanism: odixative decarboxylation of α-keto-carboxylic acid
   - energetics: irreversible
   - result:         energy released by oxidation used to generate high-energy thioester bond, reduce NAD+
- (5) succinyl-CoA synthetase
   - reaction:       succinyl-CoA  succinate [GDP + Pi  GTP;  HS-CoA]
   - enzyme:         succinyl-CoA synthetase
                                                                      Biochemistry: NOTES & OBJECTIVES (page 58 of 165)



   - mechanism: substrate level phosphorylation (and GTP + ADP  GDP + ATP)
   - energetics: reversible
- (6) succinate dehydrogenase (or Complex II of electron transport chain)
   - reaction:       succinate  fumarate [enzyme-FAD  enzyme-FADH2]
   - enzyme:         succinate dehydrogenase
   - mechanism: electrons fed directly into electron transport chain via coenzyme Q
   - energetics: irreverisble (product removal)
   - result:         oxidative generation of alkene, typical of FAD
- (7) fumarase
   - reaction:       fumarate  malate [H2O ]
   - enzyme:         fumarase
   - mechanism: hydration
   - energetics: reversible
- (8) malate dehydrogenase
   - reaction:       malate  oxaloacetate [NAD+  NADH + H+]
   - enzyme:         malate dehydrogenase
   - mechanism: dehydrogenation of secondary alcohol, forming a ketone
   - energetics: reversible
net reactions of TCA cycle
- acetyl-CoA to CO2
   - overall: acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O  2 CO2 + HS-CoA + 3NADH + 3 H+ + FADH2 + GTP
   - components:
      - acetyl-CoA  HS-CoA + 2 CO2
      - 3 NAD+  3 NADH + 3 H+
      - FAD  FADH2
      - GDP + Pi  GTP
      - 2 H2O
- pyruvate to CO2
   - overall: pyruvate + 4 NAD+ + FAD + GDP + Pi + 2 H2O  3 CO2 + 4 NADH + 4 H+ + FADH2 + GTP
   - components:
      - pyruvate  3 CO2
      - 4 NAD+  4 NADH + 4 H+
      - FAD  FADH2
      - GDP + Pi  GTP
      - 2 H2O
regulation
- isocitrate dehydrogenase
   - inhibited by: NADH
   - when NADH/NAD+ high, cell is energy rich, and ATP is not required, so TCA cycle slows
- citrate synthase
   - inhibited by: citrate, NADH, fatty acyl-CoA
   - when citrate high, cycle is full and there is no need for further oxidation to produce acetyl-CoA
   - when NADH high, there is no need for more NADH as produced in TCA cycle
   - when fatty acyl-CoA high, we will discuss later




30. Electron Transport and Oxidative Phosphorylation
Study Guide
you should understand:
- complex I: NADH dehydrogenase
   - composition: 2 redox carriers (flavin mononucleotide (FMN), iron sulfur center)
   - reaction:    NADH + H+ + CoQ  CoQH2 + NAD+
                                                                        Biochemistry: NOTES & OBJECTIVES (page 59 of 165)



   - capacity:       2 e- / molecule NADH
- complex III: ubiquinone-cytochrome c reductase
   - composition: cytochrome b, cytochrome ci, iron sulfur protein
   - reaction:       CoQH2 + 2 cyt cox  2 cyt cred + 2H+ + CoQ
   - capacity:       2 e- / molecule CoQH2
- complex IV: cytochrome oxidase
   - composition: cytochrome a, cytochrome a3, two copper centers
   - reaction:       2 cyt cred + ½ O2 + 2 H+  H2O + 2 cyt cox
   - capacity:       2 e- / molecule H2O (4 e-, or 2 NADH, to reduce one O2 molecule)
- complex II: succinate dehydrogenase
   - function:       pass e- from flavin to CoQ (unlinked to proton translocation due to small free energy)
   - reaction:       succinate + CoQ  CoQH2 + fumarate
   - capacity:       2 e- / molecule succinate
- P/O ratio: ratio of number of ATP synthesized per atom of oxygen reduced (equal to ATP / 2 e -)
   - NADHmitochondria          =3
   - NADHcytosol               = 2 (brought into mitochondria by secondary carrier, which reduces FAD)
   - FADH2                     = 2 (electrons skip complex I, and thus miss proton translocation at that point)
you should understand:
- electron carriers
   - flavin mononucleotide (FMN)
      - distribution:          I
      - capacity:              2 [H+ + e-]
   - ubiquinone (coenzyme Q, CoQ)
      - distribution:          membrane-bound accessory
      - capacity:              2 [H+ + e-]
   - cytochromes: heme prosthetic group
      - distribution:          III, IV, membrane-bound accessory
      - capacity:              1 e-
   - iron sulfur centers
      - distribution:          I, II, III
      - capacity:              1 e-
   - copper centers
      - distribution:          IV
      - capacity:              1 e-
- bucket brigade
   - electrons are passed from carrier to carrier, falling from low to high affinity
   - this is coupled with proton translocation from inside to outside the mitochondrial matrix
   - the highest affinity carrier, O2, is reduced to H2O at the end of the chain
- general pathway
   - I:    NADH + CoQ  NADH+ + H+ + CoQH2                           (proton translocation)
   - III: CoQH2 + 2 cyt cox +  CoQ + 2 cyt cred + 2 H+              (proton translocation)
   - IV: 2 cyt cred + ½ O2 + 2 H+  2 cyt cox + H2O                  (proton translocation)
   - II: succinate + CoQ  fumarate + CoQH2                          (NO proton translocation)
- ATP synthase
   - complex
      - F0: integral membrane component; contains rotating portion of enzyme
      - F1: inner surface of inner mitochondrial membrane component; catalyzes ATP formation
   - ATP formation
      - proton reentry turns part of F0 subunit, which alters physical characteristics of F1 component
      - this causes formation of ATP in 3 steps: ADP + P i  ATP  release
      - ATP generation: 3 for NADHmitochondria, 2 for FADH2 (succinate), 2 for NADHcytosol
- transport
   - ETS proton translocation create favorable gradients for incoming H +, outgoing OH-
   - ATP-4/ADP-3 translocase:             imports ADP-3, exports ATP-4 (electrogenic, driven by membrane potential)
             -    -
   - H2PO4 /OH translocase:               imports H2PO4-, exports OH- (electroneutral; driven by OH- gradient)
   - ATP synthase:                        uses H+ gradient to catalyze ATP formation
                                                                       Biochemistry: NOTES & OBJECTIVES (page 60 of 165)



  - thus it is the proton gradient and membrane potential that drive synthesis and transport


Notes: Lecture and Reading
the electron transport system
- electron transport system (ETS): overview
   - function
      - utilize reducing power of NADH, FADH2
      - convert into a form of energy that can drive ATP synthesis
   - method
      - ETS actively transports protons out of mitochondrial matrix
      - establishes an electrochemical gradient used by ATP synthase to produce ATP
   - mechanism
      - hydrogen atoms from NADH, FADH2 are separated into protons and electrons (2 [H]  2 H+ + 2 e-)
      - electrons are sequentially transferred across carriers until reaching complex IV
      - electrons are then reunited with protons to reduce molecular O 2 to H2O (2 e- + 2 H+ + ½ O2  H2O)
- composition of the electron transport chain: four membrane protein complexes
   - complex I       NADH DH:                    transfers electrons from NADH to CoQ, a membrane-bound shuttle
   - complex II succinate DH:                    transfers electrons from succinate (via FADH2) to CoQ
   - complex III ubiquinol-cyt c reductase: accepts electrons from CoQ, transfers them to cytochrome c
   - complex IV cytochrome oxidase:              accepts electrons from cytochrome c, transfers to O2 to form H2O
chemistry of the electron carriers
- flavin mononucleotide (FMN)
   - structure: active end similar to FAD
   - function: accepts 2 [H] atoms
- ubiquinone (coenzyme Q, CoQ)
   - structure: ring structure attached to hydrophobic tail composed of 10 isoprenoid units
      - isoprenoid: -CH2-CH=C(CH3)-CH2-
      - tail anchored to inner mitochondrial membrane
   - function: accepts 2 [H] atoms
- cytochromes
   - structure: proteins with heme as a prosthetic group
   - function: accepts 1 e-
- iron sulfur centers
   - structure: iron coordinated to Cys-S residues, inorganic sulfur in cubical structure
   - distribution: NADH DH complex, ubiquinol-cyt c reductase complex, succinate DH, other flavin-linked DHs
   - function: accepts 1 e-
- copper centers
   - structure: Cu coordinated to Cys-S residues, inorganic sulfur in diamond structure
   - distribution: cytochrome oxidase (complex IV)
   - function: accepts 1 e-
the affinity of electron carriers for electrons
- trend
   - during electron transport, each carrier is alternately reduced and then oxidized
   - direction of transport determined by affinity of redox carrier for electrons
   - electrons flow from good donors (low affinity; e.g. NADH) to good acceptors (high affinity; e.g. O 2)
- sequence: lowest to highest affinity
   - NADH  CI  CoQ  CIII  cytochrome c  CIV  O2
   - succinate  CII  CoQ…
energy conservation during electron transport
- net equation
      NADH + H+                NAD+ + 2 H+ + 2 e-
                   +      -
      ½ O2 + 2 H + 2 e         H2O
      ½ O2 + NADH + H+  NAD+ + H2O                        ΔG’˚ = -52.6 kcal/mol
   - large free energy release occurs in 3 sequential steps of electron transfer through I, III, IV
- complex I: NADH dehydrogenase
                                                                    Biochemistry: NOTES & OBJECTIVES (page 61 of 165)



   - composition: 2 redox carriers (flavin mononucleotide (FMN), iron sulfur center)
   - function:       pass electrons to CoQ
   - reaction:       NADH + H+ + CoQ  CoQH2 + NAD+
   - energetics: ΔG’˚ = -16.8 kcal/mol
   - capacity:       2 e- / molecule NADH
- complex III: ubiquinone-cytochrome c reductase
   - composition: cytochrome b, cytochrome ci, iron sulfur protein
   - function:       pass electrons to cytochrome c
   - reaction:       CoQH2 + 2 cyt cox  2 cyt cred + 2H+ + CoQ
   - energetics: ΔG’˚ = -9.6 kcal/mol
   - capacity:       2 e- / molecule CoQH2
- complex IV: cytochrome oxidase
   - composition: cytochrome a, cytochrome a3, two coppers
   - function:       pass electrons to and reduce O2
   - reaction:       2 cyt cred + ½ O2 + 2 H+  H2O + 2 cyt cox
   - energetics: ΔG’˚ = -25.9 kcal/mol
   - capacity:       2 e- / molecule H2O
                     - note: 4 e- to reduce one full O2 molecule
                     - thus ETS requires 2 NADH to reduce an O2 molecule
- complex II: succinate dehydrogenase
   - function:       pass e- from flavin to CoQ (unlinked to proton translocation due to small free energy)
   - reaction:       succinate + CoQ  CoQH2 + fumarate
   - energetics: ΔG’˚ = -0.9 kcal/mol
   - capacity:       2 e- / molecule succinate
coupling of ETS to ATP synthesis via proton gradient
- energy requirements of ATP synthesis
   - ADP + Pi  ATP + H2O                standard conditions:      ΔG’˚ = +7.5 kcal/mol
                                         physiological conditions: ΔG = +11 kcal/mol
   - generation of 3 ATP
      - minimum free energy requirement:          -33 kcal/mol
      - calculated ΔG’˚ for NADH to ½ O2: -52 kcal/mol
   - thus sufficient energy is available to drive the experimentally-determined ~3 ATP / NADH
      - ~1/3 of energy released over each coupling span (complex I, III, IV)
      - thus each coupling span provides sufficient energy to synthesize ~ 1 ATP / site
- coupling of electron transport to proton translocation
   - electrical potential
      - protons are pumped from mitochondrial matrix to intermembrane space
      - this generates a positive charge outside the matrix, with no compensating negative charge
   - protons at each complex
      - the number of protons brought across the membrane at each complex is not definitively known
      - complex II does not translocate protons, so FADH2 oxidation magnitude is lower than that from NADH
mechanism of ATP synthesis via oxidative phosphorylation
- ATP synthase complex
   - F0: integral membrane component; contains rotating portion of enzyme
   - F1: inner surface of inner mitochondrial membrane component; catalyzes ATP formation
- ATP formation
   - proton reentry turns part of F0 subunit, which alters physical characteristics of F1 component
      - this causes formation of ATP in 3 steps: ADP + P i  ATP  release
      - number of protons per ATP is not known with certainty, and thus we don’t know ATP / e - pair
      - by convention, we use 3 (NADH) and 2 (succinate)
   - P/O ratio: ratio of number of ATP synthesized per atom of oxygen reduced
      - equivalent to number of ATP synthesized per pair of e- entering the chain
      - P/ONADH = 3
      - P/OFAD = 2 (skips complex I)
- cytosolic NADH
   - NADH from cytosol cannot cross mitochondrial membrane to be directly oxidized by ETS
                                                                       Biochemistry: NOTES & OBJECTIVES (page 62 of 165)



   - instead, used to reduce molecules that can enter the mitochondria
      - this will in turn reduce FAD or NAD+ in the matrix
      - depending on this, cytosolic NADH will yield 2 or 3 ATP
ATP, ADP, and Pi transport
- ADP levels in mitochondria are dependent on amount of ATP use in cytoplasm
   - ATP synthesized in mitochondria:            transported to cytoplasm for utilization
   - ADP, Pi formed from hydrolysis:             transported to mitochondria for ATP synthesis
- ATP/ADP transport
   - transport: based on a single electrogenic ATP/ADP transporter/exchange system
      - electrogenic: creates an electrical potential
      - 4 negative charges (ATP-4) brought out, but only 3 (ADP-3) brought in
   - interior negative membrane potential thus drives ejection of ATP, coupled uptake of ADP
- phosphate transport
   - transport: based on Pi/OH- antiporter (OH- out, Pi in)
   - alkaline interior of mitochondria drives in P i against concentration gradient
- energetics of transport
   - everything is coupled
   - energy from e- transport stored in proton gradient, membrane potential, also drives transport, ATP synthesis




31. Control of Oxidative Phosphorylation and Glucose Oxidation
Study Guide
you should understand:
- poisons
   - dinitrophenol: uncoupler that shuttles protons across membrane
      - H+ gradient:         decreased         (enzyme action)
      - ATP synthesis:       decreased         (proton gradient destroyed)
      - electron transport: increased          (absence of negative feedback)
      - oxygen use:          increased         (coupled to ETS)
      - NADH/NAD+:           decreased         (increased ETS uses more NADH)
      - ATP in matrix:       decreased         (ATP not made from ADP)
   - cyanide: inhibits ETS (cytochrome oxidase)
      - H+ gradient:         decreased         (decreased ETS)
      - ATP synthesis:       decreased         (proton gradient destroyed)
      - electron transport: decreased          (enzyme action)
      - oxygen use:          decreased         (coupled to ETS)
      - NADH/NAD+            increased         (decreased ETS uses less NADH)
      - ATP in matrix:       decreased         (ATP not made from ADP)
   - bongkrekic acid: inhibits ATP/ADP translocase
      - H+ gradient:         increased         (exported protons unable to return)
      - ATP synthesis:       decreased         (no substrate)
      - electron transport: decreased          (coupled to ATP synthesis)
      - oxygen use:          decreased         (coupled to ETS)
      - NADH/NAD+            increased         (decreased ETS uses less NADH)
      - ADP in matrix:       decreased         (ADP converted to ATP, unable to leave)
- feedback inhibition
   - respiratory control: ADP
      - when ADP is lacking (due to cellular presence as ATP), protons cannot reenter cell (slowed ATP synthase)
      - when protons cannot reenter, proton gradient builds up (slowed electron transport)
   - causal relationships: high ATP/ADP  high NADH/NAD+  increased citrate, acetyl-CoA
   - feedback inhibition
      - TCA cycle
                                                                      Biochemistry: NOTES & OBJECTIVES (page 63 of 165)



       - citrate synthase:         inhibited by NADH, citrate
       - isocitrate DH:            inhibited by NADH
       - pyruvate DH:              inhibited by NADH, acetyl-CoA
    - glycolysis
       - pyruvate kinase:          inhibited by ATP
       - phosphofructokinase:      inhibited by ATP, activated by AMP
       - hexokinase:               inhibited by glucose 6-phosphate


Notes: Lecture and Reading
the rate of electron transport is coupled to ATP synthesis and disruption by poisons
- oxidative phosphorylation is tightly coupled to the electron transfer process
   - coupling: one process requires the other
   - experiment: [O2] vs. time
      - setup
         - isolated mitochondria depleted of ADP are incubated in buffer (which includes P i)
         - concentration of O2 in solution is measured with an O2 electrode
      - observations
         - in absence of ADP, decline in [O2] is negligible
         - upon addition of ADP, [O2] declines heavily, but slows after all ADP is used
         - further addition of ADP once again causes O2 to decline
      - conclusion: electron transport (measured via [O2] vs. time) requires ADP
   - experimental parameters
      - P/O ratio: determine amount of ATP synthesized upon addition of ADP, read O 2 consumed off graph
      - respiratory control ratio: rate of O 2 use (with ADP)
                                    rate of O 2 use (without ADP)
         - equal to sharp slope over relaxed slope
         - properly-prepared mitochondria can have this as high as (10/1)
- DNP uncouples electron transport from ATP synthesis
   - dinitrophenol (DNP): “uncoupler” of electron transport and phorphorylation
      - uncoupler: lipid-soluble weak acids that carry H+ back and forth across membrane (approach equilibrium)
      - ATP synthase: does not work (ETS cannot establish electrochemical potential)
      - ETS: works at maximal velocity (back pressure of proton gradient never builds up
   - experiment: [O2] vs. time
      - setup: DNP added to rapidly phosphorylating mitochondria
      - observations: O2 is rapidly used up, but no ATP is made
   - conclusion: DNP uncouples electron transport from ATP synthesis
- naturally uncoupled mitochondria / thermogenesis
   - shivering: small muscular contractions that dissipate metabolic energy as heat
   - nonshivering thermogenesis: use of uncoupled mitochondria in brown fat to generate heat
      - signal from sympathetic nervous system responds to cold shock, unmasks H+ channel
      - NADH oxidation is uncoupled from ATP production, giving a rapid production of heat
      - distribution: neonatal tissue in humans, hibernating animals, small animals
- inhibitors / poisons
   - cyanide: inhibits cytochrome oxidase of the ETS
   - bongkrekic acid: inhibits ATP/ADP translocase
ATP synthesis is regulated by cellular energy demands
- regulation of oxidative phosphorylation by ADP levels is an important physiological control
   - respiratory control in cells with normal, coupled ATP synthase and ETS
      - low ADP signals that most is in ATP form
      - ATP synthesis slows, protons cannot reenter matrix, electrochemical gradient builds up, and ETS slows
   - ATP/ADP: indicator of cellular energy status
      - with little ADP, decreased mitochondrial [ADP] indicates plenty of ATP in cytoplasm
      - as such, it is wasteful for cells to continue to oxidize nutrients, since little more ATP can be made
   - AMP concentration: measure of ATP/ADP ratio
                                                                    Biochemistry: NOTES & OBJECTIVES (page 64 of 165)



      - adenylate kinase: ATP + AMP ↔ ADP + ADP (Keq = 1)
      - Keq = 1 = [ADP]2 / [ATP]∙[AMP], where [AMP] = [ADP]2/[ATP]
      - [AMP] increases as a function of [ADP]2, making AMP a very sensitive indicator of low ATP (higher ADP)
- regulation of glucose utilization and oxidation in the muscle
   - ATP demands in skeletal muscle
      - vary widely with changes in rates of muscle contraction
      - [ATP] < 6 mM in muscle cells
      - thus ATP must be synthesized at a rate appropriate to muscle contraction
   - depleted ATP: use of phosphocreatine as a reservoir for high energy phosphate
      - with decreasing ATP, a kinase catalyzes:
            phosphocreatine  creatine [creatine kinase: ADP  ATP]
            - reversible (ΔG’˚ of hydrolysis = -10 kcal/mol)
            - stabilization: creatine has resonance delocalization, phosphocreatine does not
      - phosphocreatine concentrations ~20-30 mM, thus increasing high energy phosphate content 5-fold
      - creatinine: breakdown product that is excreted via kidney
   - depleted ATP, phosphocreatine: rely on oxidation of carbon sources like glucose
- rate of oxidative phosphorylation: controls rate of fuel oxidation by feedback allosteric regulation
   - high/low NADH
      - if ATP is high, [ADP] is low, limiting rate of phosphorylation and ETS, leading to buildup of NADH
      - in absence of other controls, NAD+ would be depleted, preventing function of TCA cycle
      - however, NADH allosterically inhibits pyruvate DH of TCA cycle, preventing NAD + depletion
      - when speaking of elevated NADH, we are actually speaking of high NADH/NAD +
   - simple relationships: high ATP/ADP  high NADH/NAD+  high citrate, acetyl-CoA
   - key enzymes, allosteric regulators of glucose oxidation
      - relationships
         - NADH: regulates mitochondrial enzymes
         - ATP, AMP: regulates cytoplasmic enzymes
      - regulation
         - isocitrate dehydrogenase:              inhibited by NADH
         - citrate synthase:                      inhibited by NADH, citrate
         - pyruvate dehydrogenase:                inhibited by NADH, acetyl-CoA
         - pyruvate kinase:                       inhibited by ATP
         - phosphofructokinase:                   inhibited by ATP, activated by AMP
         - hexokinase:                            inhibited by glucose 6-phosphate
   - reverse cascade: slowing glucose oxidation
      - TCA cycle
         - high NADH: inhibits isocitrate DH, resulting in accumulation of citrate
         - high citrate, NADH: inhibits citrate synthase, slowing entry of acetyl-CoA into TCA cycle
         - high acetyl-CoA, NADH: cause phosphorylation, inactivation of pyruvate DH
      - glycolysis
         - high ATP: inhibits pyruvate kinase
         - high ATP: inhibits phosphofructokinase; high AMP: activates it
         - high glucose 6-phosphate: inhibits hexokinase
- lactic acid production in muscle during anaerobic exercise
   - decreased O2 availability leads to an increased NADH/NAD+
   - this leads to low ATP, high AMP, which promotes rapid glucose metabolism via glycolysis
   - lactic acid: formed from pyruvate; reaction regenerates NAD +




32. Glycogen Metabolism
Study Guide
you should understand:
                                                                        Biochemistry: NOTES & OBJECTIVES (page 65 of 165)



- glycogen structure
   - hemiacetal glucose ring
      - 6 carbon ring formed by C5 –OH reaction with C1 =O, forming –O– linkage between C1 and C5
      - C1: anomeric carbon (achiral carbon made into a chiral center by ring formation)
         - α form: OH faces down
         - β form: OH faces up
      - C2, C3, C4: OH faces down, up, down
   - linkage
      - α(1,4): α-D-glucose C1 reacts with C4 of another glucose, forming glycosidic bond (typical of straight chains)
      - α(1,6): α-D-glucose C1 reacts with C6 of another glucose, forming glycosidic bond (typical of branch points)
   - glycogen: large, branched polymer of α-D-glucose
      - linear portion:       α-(1,4)-glycosidic linkages (93% of glucosyl residues)
      - branches:             α-(1,6)-glycosidic linkages (7% of glucosyl residues)
      - glycogen polarity
         - non-reducing ends:          free C4 –OH, point of synthesis (tree branches)
         - reducing end:               free C1 –OH; root point of glycogen (tree trunk)
- glycogen degradation
   - glycogen phospohrylase: glucosyln+1 + H3PO4  glucosyln + glucose 1-phosphate
      - irreversible: high H3PO4, low glucose 1-phosphate
   - mutase: glucose 1-phosphate  glucose 6-phosphate
      - muscle:      phosphofructose isomerase, entry into glycolysis
      - liver:       glucose 6-phosphatase, release into bloodstream
   - debranching enzyme: transfers terminal 3 glucosyl residues of a 4 glucosyl branch to another non-reducing end
   - α(1,6)-glucosidase: hydrolyzes branch point to yield free glucose
- glycogen synthesis
   - hexokinase: glucose  glucose 6-phosphate
   - mutase: glucose 6-phosphate  glucose 1-phosphate
   - UDP-glucose pyrophosphorylase: glucose 1-phosphate  UDP glucose + PPi
      - pyrophosphatase: PPi  2 Pi
   - glycogen synthase: UDP glucose + glucosyln  glucosyln+1 + UDP
      - irreversible: high UDP-glucose, low UDP
   - UDP + ATP  UTP + ADP


Notes: Lecture and Reading
hemiacetal form of glucose
- forms of glucose
   - aldehyde/ketone form:             linear Fischer structure              < 1% in solution
   - hemiacetal/hemiketal form:        ring      Haworth structure           > 99% in solution
- hemiacetal form of glucose
   - anomeric carbon: imparts another asymmetric carbon on glucose
      - α form: OH up in Haworth structure
      - β form: OH down in Haworth structure
   - glucose distribution at equilibrium
      - α form:               36 %
      - β form:               63 %
      - carbonyl form:        1%
structure and function of glycogen
- large, branched polymer of α-D-glucose
   - linear portion: α-(1,4)-glycosidic linkages (93% of glucosyl residues)
   - branches:       α-(1,6)-glycosidic linkages (7% of glucosyl residues)
- function: readily mobilized form of glucose
   - structural logic
      - more efficient: less water to dissolve, less osmotically active than would be normal glucose
      - highly branched: makes rapid degradation easier
   - tissue distribution
                                                                      Biochemistry: NOTES & OBJECTIVES (page 66 of 165)



      - muscle: glucose 6-P used to drive muscle contraction
      - liver: used as a glucose reserve to maintain blood glucose levels
- glycogen polarity
   - non-reducing ends:        4-OH; where synthesis and degradation occurs
   - reducing end:             1-OH; point from which all others branch
glycogen degradation
- glycogen phosphorylase
   - reaction:       glucosyln+1 + H3PO4  glucosyln + glucose 1-phosphate
   - enzyme:         glycogen phosphorylase
   - mechanism: phosphorolysis
                     - conserves some energy of glycosidic bond in hemiacetal phosphate
                     - hydrolysis (to glucose) would have required ATP use to make G6P
   - energetics: irreversible
                     - ΔG’˚ = +1 kcal/mol, but high Pi, low G1P drives reaction
                     - G1P is low due to ready conversion to G6P
- mutase
   - reaction:       glucose 1-P  glucose 6-P
   - enzyme:         mutase
   - mechanism: intramolecular rearrangement (mutation)
                     - cleavage of hemiacetal bond (ΔG’˚ = 5kcal/mol)
                     - formation of phosphoester bond (ΔG’˚ = 3 kcal/mol)
   - energetics: reversible
- fate of glucose 6-P
   - muscle: proceeds through glycolysis to supply ATP
   - liver: hydrolysis to glucose (glucose 6-phosphatase), release into bloodstream
- glycogen degradation summary
   - glycogen phosphorylase:                      produces glucose 1-phosphate, shortens glycogen chain by 1
   - mutase:                                      rearranges glucose 1-phosphate to glucose 6-phosphate
   - glucose 6-phosphatase (liver):               hydrolyzes glucose 6-phosphate, sends glucose to blood
   - phosphoglucose isomerase (muscle):           enters glucose 6-phosphate into glycolysis
- debranching
   - glycogen phosphorylase falls off when glucosyl chain length is reduced to 4
   - debranching enzyme transfers terminal three glucosyl residues to C4 of glucosyl residue of another branch
   - α-(1,6)-glucosidase hydrolyzes branch point to yield free glucose
      - α-(1,6) linkage: hydrolyzed into glucose                    glycolysis  2 ATP
      - α-(1,4) linkage: phosphorylized into glucose 6-P            glycolysis  3 ATP
glycogen synthesis
- regulation
   - degradation: low ATP, blood glucose
   - synthesis: high ATP, blood glucose
- formation of glucose 1-phosphate
   - glucose  glucose 6-phosphate [hexokinase]
   - glucose 6-phosphate  glucose 1-phosphate [mutase]
- formation of UDP-glucose
   - energetics
      - hydrolysis of G1P:                        ΔG’˚ = -5 kcal/mol
      - formation of glycosidic bond:             ΔG’˚ = 4 kcal/mol
      - though this is sufficient in standard state, low [G1P] necessitates additional activation
      - this is done via conversion to UDP-glucose
   - UDP-glucose pyrophosphorylase
      - reaction: glucose 1-phosphate + UTP  UDP-glucose [ (PPi  2 Pi) ]
      - enzyme: UDP-glucose pyrophosphorylase
      - energetics: irreversible (pyrophosphatase and hydrolysis of PP i drives)
- glycogen synthase: elongation
   - reaction:       UDP-glucose + glucosyln  glucosyln+1
   - enzyme:         glycogen synthase
                                                                       Biochemistry: NOTES & OBJECTIVES (page 67 of 165)



   - mechanism: - cleavage of hemiacetal bond (ΔG’˚ = -5 kcal/mol)
                     - formation of glycosidic bond (ΔG’˚ = +4 kcal/mol)
   - energetics: irreversible
                     - high UDP-glucose, low UDP
                     - UDP  UTP (dinucleotide kinase), drives reaction by mass action
- glycogen synthesis summary
   - mutase:                                    glucose 6-phosphate  glucose 1-phosphate
   - UDP-glucose pyrophosphorylase:             glucose 1-phosphate  UDP glucose
   - glycogen synthase:                         UDP glucose + glycogen  glycogenn+1 + UDP
   - diphosphonucleotide kinase:                converts UDP to UTP (ATP  ADP)
- branching
   - branching enzyme
      - transfers oligoglucans (~7 long) from growing linear chain of >11
      - attached to C6 of a glucosyl residue at least 4 residues away
   - doubles non-reducing ends, and elongation and branching result in the final glycogen molecule




33. Regulation of Glycogen Metabolism
Study Guide
you should understand:
- adrenalin response
   - adrenalin (epinehprine): released from adrenal medulla into blood in response to stress stimuli
   - muscle: readies muscle for immediate action, maintains the stimulated muscle response
   - non-muscle: increases heart rate, promotes vasoconstriction
- adrenalin-stimulated increase of cAMP, a second messenger
   - adrenalin reversibly binds receptor, causing conformational change in effector domain
   - Gs(αβγ) complex binds effector domain, releasing GDP and binding GTP from α subunit
   - Gsα dissociates from Gs complex, binds activation domain of adenylate cyclase
      - adenylate cyclase: ATP  3’,5’-cyclic-AMP (cAMP) + PPi
      - cAMP: intracellular second messenger of adrenalin binding
- cAMP-mediated activation of glycogen phosphorylase, inhibition of glycogen synthase
   - 4 cAMP binds protein kinase A, releasing the two regulatory subunits (R 2) from the two catalytic subunits (C2)
   - protein kinase A phosphorylates and inactivates glycogen synthase, slowing glycogen synthesis
   - protein kinase A phosphorylates and activates phosphorylase kinase
   - phosphorylase kinase phosphorylates and activates glycogen phosphorylase, speeding glycogen degradation
- reversing the adrenalin signal
   - protein phosphatases
      - constitutive dephosphorylation of PKA, phosphorylase kinase, glycogen synthase
      - returns the glycogen proteins to steady state mode (synthesis)
   - allosteric regulation
      - glycogen synthase-P (inactive): activated by high glucose 6-P (indicating high glucose)
      - glycogen phosphorylase (inactive): activated by high 5’ AMP (indicating lack of glucose)
   - signal termination
      - adrenalin: low concentrations cause it to fall off β-adrenergic receptor
      - GTPase of Gsα
         - hydrolyzes GTP to GDP, causing dissociation from adenylate cyclase
         - GDP-Gsα binds βγ, reforming the Gs(αβγ) complex
      - cAMP phosphodiesterase: hydrolyzes cAMP into 5’AMP
- adrenalin must be constitutively present for maintenance of the signal
- protein kinase A: cAMP-dependent activation
   - structure: R2C2 (R = regulatory inhibition, C = catalytic)
      - R2C2 (inactive) + 4 cAMP  2 R-cAMP2 + 2 C (active)
                                                                      Biochemistry: NOTES & OBJECTIVES (page 68 of 165)



      - two cAMP must bind to dissociate a single R (total of 4 cAMP for activation)
   - function: phosphorylation of Ser and Thr residues in target proteins
      - serves to regulate activity of enzymes
      - ATP: used in phosphorylation (kinase) reaction
- protein kinase A-dependent regulation of glycogen enzymes
   - glycogen synthase: directly phosphorylated (inactivated)
   - glycogen phosphorylase
      - phosphorylase kinase: phosphorylated (activated) by PKA
      - glycogen phosphorylase: phosphorylated (activated) by phosphorylase kinase
   - phosphatase: reverses phosphorylations of these enzymes, returning molecules to steady synthetic state
- additional enzymatic regulation
   - glycogen synthase-P: allosterically activated by glucose 6-P
      - false alarms: glucose formed by glycogen breakdown turns out to be unnecessary
      - as such, it becomes necessary to reconstitute glycogen, even with high adrenalin still in blood
   - glycogen phosphorylase: allosterically activated by 5’AMP
      - conditions of maximal muscle contraction
      - further increases amount of glucose 6-P available for glycolysis


Notes: Lecture and Reading
regulation by adrenalin
- adrenalin (epinehprine): released from adrenal medulla into blood in response to stress stimuli
   - initiation: neural stimulation of adrenal medulla
   - stress stimuli: excitement/anticipation, fear, “fight or flight”
- adrenaline response
   - tissue
      - muscle: readies muscle for immediate action, maintains the stimulated muscle response
      - non-muscle: increases heart rate, promotes vasoconstriction
   - mechanism: overview
      - adrenalin binds β-adrenergic receptor, which ultimately increases intracellular 3’,5’-cyclic-AMP (cAMP)
      - cAMP activates protein kinase A
      - this ultimately activates glycogen phosphorylase and inhibits glycogen synthase
mechanism of adrenalin-stimulated increase in cAMP
- components
   - adrenalin: blood concentration varies with sympathetic adrenal stimulation
   - β-adrenergic receptor: IMP that binds, transmits adrenaline signal
   - Gs complex: GTP-binding heterotrimer (α,β,γ)
   - adenylate cyclase: enzyme responsible for ATP  cAMP
   - intracellular signal: molecule that serves as a second messenger
- mechanism
   - adrenalin reversibly binds receptor, proportional to blood adrenalin concentration
   - effector domain of receptor undergoes conformational change, binds Gs complex
   - Gsα releases GDP, binds GTP, and dissociates from Gs complex
   - Gsα binds adenylate cyclase, activating the enzyme
   - adenylate cyclase catalyzes ATP  3’,5’-cyclic-AMP (cAMP) + PPi
   - cAMP serves as the intracellular signal
- turnover of cAMP
   - cAMP phosphodiesterase: hydrolyzes cAMP into 5’-AMP
   - thus [cAMP] reflects both rate of synthesis and degradation
- recycling of Gsα
   - Gsα GTPase: hydrolyzes bound GTP to GDP, which causes dissociation from adenylate cyclase
   - Gsα returns to β,γ complex
   - for continuous activation of adenylate cyclase, adrenaline must continue to bind receptor
cyclic AMP-mediated activation of glycogen phosphorylase, inhibition of glycogen synthase
- protein kinase A: cAMP-dependent activation
   - structure: R2C2 (R = regulatory inhibition, C = catalytic)
                                                                         Biochemistry: NOTES & OBJECTIVES (page 69 of 165)



      - R2C2 (inactive) + 4 cAMP  2 R-cAMP2 + 2 C (active)
      - two cAMP must bind to dissociate a single R (total of 4 cAMP for activation)
   - function: phosphorylation of Ser and Thr residues in target proteins
      - serves to regulate activity of enzymes
      - ATP: used in phosphorylation (kinase) reaction
- protein kinase A-dependent regulation of glycogen enzymes
   - glycogen synthase: directly phosphorylated (inactivated)
   - glycogen phosphorylase
      - phosphorylase kinase: phosphorylated (activated) by PKA
      - glycogen phosphorylase: phosphorylated (activated) by phosphorylase kinase
   - phosphatase: reverses phosphorylations of these enzymes, returning molecules to steady synthetic state
- additional enzymatic regulation
   - glycogen synthase-P: allosterically activated by glucose 6-P
      - false alarms: glucose formed by glycogen breakdown turns out to be unnecessary
      - as such, it becomes necessary to reconstitute glycogen, even with high adrenalin still in blood
   - glycogen phosphorylase: allosterically activated by 5’AMP
      - conditions of maximal muscle contraction
      - further increases amount of glucose 6-P available for glycolysis
glycogen metabolism in the liver: preview
- adrenalin: regulates degradation, synthesis using same mechanism shown here
- glucagon: stimulates glycogenolysis using a similar mechanism to activate adenylate cyclase
   - muscle lacks glucagon receptors
   - this gives liver additional ability to release sugar when blood sugar is low
- insulin: promotes glycogen synthesis in both liver and muscle
glycogen storage diseases
- glycogen storage diseases: deficiencies in glycogen metabolism
- cause: inherited defects in enzymes of glycogen metabolism
- effects: poor glycogen metabolism in liver, sometimes also in skeletal muscle




34. Sources of Glucose, Other Auxiliary Paths of Sugar Metabolism
Study Guide
you should understand:
- galactose metabolism
   - enzymes
      - galactokinase:         galactose  galactose 1-P [ATP  ADP]
      - UDP-transferase: galactose 1-P  UDP galactose [UDP glucose  glucose 1-P]
      - epimerase:             UDP galactose  UDP glucose
      - (UDP glucose  glucose 1-P via UDP transferase coreaction)
   - defects: galactosemia
      - kinase: galactose accumulates, is converted to galacticol in some cells, especially eyes where it forms cataracts
      - UDP transferase: galactose and galactose 1-P accumulate, and are toxic to cells
         - untreated: mental retardation, liver failure, death
         - galactose-free diet: long-term complications (speech ataxia, ovarian dysfunction, neurological defects)
         - reason for long term complications is unknown
      - epimerase: accumulation of UDP-galactose, causing similar problems as UDP-transferase
- aldose reductase
   - function: production of sorbitol, useful to certain tissues
   - side reactions
      - glucose  sorbitol [NADPH + H+  NADP+]  fructose [NAD+  NADH + H+, slow]
      - galactose  galacticol [NADPH + H+  NADP+, metabolic dead end]
   - sorbitol and galacticol accumulate, cause osmotic swelling, and give associated pathologies
                                                                       Biochemistry: NOTES & OBJECTIVES (page 70 of 165)



- pentose phosphate pathway
   - glucose 6-P  6-phosphogluconolactone [glucose 6-P dehydrogenase: NADP+  NADPH + H+]
   - 6-phosphogluconolactone  6-phosphogluconate [ H2O]
   - 6-phosphogluconate  ribulose 5-phosphate [6-phosphogluconate DH: NADP+  NADPH + H+,  CO2]
   - ribulose 5-phosphate  ribose 5-phosphate [isomerase]
- lactose utilization
   - lactose: disaccharide composed of galactose β(14) linked to glucose
   - lactase: enzyme in brush border of intestinal enterocytes responsible for digestion, absorption of lactate
      - lactase is lost with age, often causing lactose intolerance in older people
      - diarrhea due to osmotic imbalance, flatulence due to digestion by normal flora
      - rare genetic defect in lactase also exists
- disaccharides of the human diet
   - maltose: glucose to glucose in α(14) linkage reducing
   - lactose: galactose to glucose in β(14) linkage reducing
   - sucrose: glucose to fructose in α1β2 linkage non-reducing


Notes: Lecture and Reading
dietary sources of glucose and other sugars
- sugars
   - monosaccharides (glucose, fructose)
      - found in honey and some kinds of fruit
      - can be absorbed directly by intestinal mucosal cells
   - disaccharides, polysaccharides (e.g. starch)
      - form the bulk of sugar intake
      - must be hydrolyzed prior to absorption
   - reducing vs. non-reducing
      - reducing: aldehyde/ketone group at anomeric carbon available for oxidation
      - non-reducing: no available anomeric aldehyde/ketone group
   - disaccharides common in human diet
      - maltose:               2 x α-D-glucose                     α(14) glycosidic bond               reducing
      - lactose:               β-D-galactose, α-D-glucose          β(14) glycosidic bond               reducing
      - sucrose:               α-D-glucose, β-D-fructose           α1β2 glycosidic bond                non-reducing
- digestion
   - digestive enzymes
      - salivary, pancreatic amylase: randomly attack α(14) bonds of starches to give maltose, isomaltose, glucose
      - disaccharidases: hydrolyze sucrose, lactose, maltose into monosaccharides
         - sucrase: cleaves sucrose into glucose, fructose
         - lactase: cleaves lactate into glucose, galactose
         - maltase: cleaves maltose into 2 glucose
   - intestinal mucosal cells absorb monosaccharides
AUXILIARY PATHWAYS OF SUGAR METABOLISM
lactose metabolism
- lactase: found on brush border of intestinal epithelial cells
   - declines in activity with age, and adults often have decreased ability to digest
   - during evolution, milk was not normally encountered in adult diet
- lactose intolerance: condition where milk products are not properly digested
   - intestinal flora flourish, cause gas; osmotically-active sugar hinders intestinal water absorption
   - treatment: avoid milk products; medication (lactase enzyme)
   - note: there is a genetic defect unrelated to the age-dependent decline
galactose metabolism
- pathway
   - galactokinase:            galactose  galactose 1-P (ATP  ADP)
   - UDP transferase:          galactose 1-P  UDP galactose (UDP-glucose  glucose 1-P)
   - epimerase:                UDP galactose  UDP glucose
   - net result:               conversion of galactose to glucose 1-P, using 1 ATP
                                                                         Biochemistry: NOTES & OBJECTIVES (page 71 of 165)



- galactosemia: human deficiencies in galactose metabolism
   - galactokinase defects
      - mechanism:             galactose is not phosphorylated, and accumulates
                               - in some cells, converted to galacitol by aldose reductase
      - symptoms:              cataracts (galacitol accumulation in eye, causing osmotic disturbances)
      - treatment:             galactose-free diet
   - UDP transferase defects
      - mechanism:             galactose and galactose 1-P accumulation
      - symptoms:              mental retardation, liver failure, death
      - treatment:             galactose-free diet
                               - long-term complications occur (speech ataxia, ovarian dysfunction, retardation)
                               - cause of long term complications is unknown
   - epimerase defects: similar to UDP transferase defects
- metabolism with aldose reductase
   - aldose reductase
      - reaction: glucose  sorbitol [NADPH + H+  NADP+]
                     galactose  galacitol [NADPH + H+  NADP+]
      - enzyme: aldose reductase
   - accumulation
      - galacitol: metabolic dead end
      - sorbitol (glucitol): can be converted to fructose in a slow NAD + dependent reaction, but still accumulates
   - consequences: swelling of the cell, pathological damage (e.g. cataracts)
pentose phosphate pathway
- overall: glucose 6-phosphate + 2 NADP+ + H2O  ribose 5-phosphate + 2 NADPH + 2 H+ + CO2
- significance
   - forms pentoses for nucleotide formation
   - source of NADPH for biosynthesis, reduction of molecules like oxygen radicals or glutathione
- pathway
   - glucose 6-P  6-phosphogluconolactone
      - enzyme: glucose 6-P dehydrogenase
      - coreactions: NADP+  NADPH + H+
      - energetics: rate-limiting, regulated step
   - 6-phosphogluconolactone  6-phosphogluconate [H2O]
   - 6-phosphogluconate  ribulose 5-phosphate
      - enzyme: 6-phosphogluconate dehydrogenase
      - coreactions: NADP+  NADPH + H+;  CO2
   - ribulose 5-phosphate  ribose 5-phosphate
- glucose 6-phosphate DH: clinical significance
   - some individuals genetically lack this enzyme in erythrocytes or have an impairment
   - unable to maintain normal NADPH for biosynthesis
   - this makes RBCs more susceptible to hemolysis




35. Reactive Oxygen Species and Oxidative Stress
Study Guide
you should understand:
- reactive oxygen species: chemistry
   - O2-       superoxide anion
   - HOOH hydrogen peroxide
   - ●OH       hydroxyl radical
   - sequential electron additions: O2  O2-  HOOH  ●OH  H2O
- generating reactive oxygen species in vivo
                                                                       Biochemistry: NOTES & OBJECTIVES (page 72 of 165)



   - superoxide: O2 + e-  O2-
      - incomplete ETS: CoQH2 + O2  CoQH● + O2-
      - methemoglobin: Hb(Fe2+) + O2  Hb(Fe3+) + O2-
   - hydrogen peroxide
      - FAD-linked oxidases: FADH2 + O2  FAD + H2O2
      - superoxide dismutase: 2 H+ + 2 O2-  H2O2 + O2
   - hydroxyl radicals
      - Fenton reaction: HOOH  OH- + ●OH [nonenzymatic: Fe2+  Fe3+]
- phagocytosis and respiratory burst oxidase
   - respiratory burst oxidase: catalyzes the formation of superoxides used in response to infection
      - reaction: 2 O2  2 O2- + 2 H+ [RBO: NADPH  NADP+]
      - also promotes formation of other ROS (e.g. HOOH by superoxide dismutase)
   - regulation: activated in response to microbial infection
   - defects: Chronic Granulomatous Disease; affects immunity
- damage by ROS
   - nucleic acids (●OH): strand breakage, base damage/elimination
   - proteins
      - O2-     inactivation of Fe-S centers
      - H2O2 oxidation of –SH groups
      - ●OH abstraction of hydrogen atoms
   - lipids (●OH): abstraction of hydrogen atoms leading to lipid peroxidation, decrease in fluidity
- antioxidant enzymes
   - superoxide dismutase
      - reaction: 2 H+ + 2 O2-  H2O2 + O2
      - function: detoxification of superoxide radicals, formation of peroxides (mitochondria, cytoplasm)
   - catalase
      - reaction: 2 HOOH  2 H2O + O2
      - function: detoxification of hydrogen peroxide (peroxisomes, mitochondria)
   - glutathione peroxidase
      - reaction: 2 GSH + HOOH  GS-SG +2 H2O
                     2 GSH + ROOH  GS-SG + H2O + ROH
      - function: detoxificaiton of hydrogen peroxide and other peroxides, esp. lipids
      - regeneration: GS-SG  2 GSH [glutathione reductase: NADPH + H+  NADP]
- repair pathways
   - damage
      - Hb(Fe2+) + O2  Hb(Fe3+) + O2- [spontaneous]
      - 2 O2- + 2 H+  HOOH + O2 [superoxide dismutase]
      - HOOH  ●OH [Fenton reaction; Fe2+  Fe3+]
   - repair
      - proteins:             degradation through proteosomes
      - lipids:               peroxidation
      - nucleic acids:        NER and other pathways
   - removal: peroxides
      - 2 GSH + HOOH  GS-SG + H2O [glutathione peroxidase]
      - GS-SG  2 GSH [glutathione reductase: NADPH + H+  NADP+]
      - glucose 6-P  6-phosphogluconolactone [glucose 6-P DH: NADP+  NADPH + H+]


Notes: Lecture and Reading
reactive oxygen species
- molecular oxygen: two unpaired electrons, and thus fairly reactive
- cytochrome oxidase: reduction of oxygen by sequential electron addition
   - reaction:   O2 + 4 H+ + 4 e-  2 H2O
   - enzyme:     cytochrome oxidase
   - mechanism: sequential addition of electrons
                 - small portion of O2 is not completely reduced
                                                                          Biochemistry: NOTES & OBJECTIVES (page 73 of 165)



                     - consequently, 3 reactive oxygen species (ROS) arise
- electron chemistry of oxygen
   - O2 (+ e-)  O2- (+ e-)  H2O2 (+ e-)  OH- + ●OH (+ e-)  H2O
   - reactions reflect 1 e- transfers, but do not reflect balanced equations
- reactive oxygen species
   - superoxide anion:         O-O-               one unpaired electron, moderately reactive
                                                  - acts as a single electron-oxidizing agent
   - hydrogen peroxide:        HOOH               no unpaired electrons, relatively stable
                                                  - oxidizes –SH residues on proteins
                               ●
   - hydroxyl radical:           OH               one unpaired electron, most reactive ROS
                                                  - abstracts H atom or adds on to existing molecule
                                                  - short half life in solution
generation of reactive oxygen species during normal metabolism
- superoxide radicals (O2-)
   - reaction: O2 + e-  O2- (one electron transfer)
   - electron sources
      - mitochondria: normal metabolism
         - inadvertent transfer of an electron to O2
         - reaction: CoQH2 + O2  CoQH● + O2-
      - red blood cells: spontaneous reduction of O2 bound to Hb
         - reaction: Hb(Fe2+) + O2  MetHb(Fe3+) + O2-
         - repair: 2 MetHB(Fe3+)  2 Hb(Fe2+) [methemoglobin reductase: NADH  NAD+]
         - methemoglobin: result of spontaneous O2 reduction, Fe2+ oxidation
         - methemoglobin reductase: reduces methemoglobin to hemoglobin
- hydrogen peroxide (H-O-O-H)
   - product of some FAD-linked oxidase reactions (e.g. xanthine oxidase)
   - product of catalase enzyme in peroxisomes, mitochondria
- hydroxyl radicals (●OH)
   - Fenton reaction: spontaneous reduction of HOOH in the presence of iron(2)
   - reaction: HOOH  ●OH + OH- [Fe2+  Fe3+]
elimination of reactive oxygen species
- antioxidants
   - vitamin E: lipid-soluble vitamin that acts to trap free radicals
      - prevents peroxidation of fatty acids in adipose tissue, biological membranes
      - deficiencies are rare, symptoms include muscular weakness and fragile RBCs
      - more important with additional dietary intake of polyunsaturated fatty acids
   - vitamin A: lipid-soluble vitamin that sequesters ROS
   - vitamin C: water-soluble vitamin that keeps vitamin E, vitamin A in reduced states
- antioxidant enzymes
   - superoxide dismutase: reduction of superoxide
      - reaction:              2 H+ + 2 O2-  H2O2 + O2
      - enzyme:                superoxide dismutase (SOD)
      - distribution:          MnSOD mitochondria
                               Cu/Zn SOD: cytoplasm
   - catalase: reduction of peroxide
      - reaction:              H2O2 + H2O2  O2 + 2 H2O
      - enzyme:                catalase
      - distribution:          peroxisomes, mitochondria
   - glutathione peroxidase: reduction of peroxide
      - reaction:              2 GS-H + H2O2  GS-SG + 2 H2O
                               2 GS-H + ROOH  GS-SH + HOH + ROH
      - enzyme:                glutathione peroxidase
      - distribution:          primary enzymatic system for control of cellular peroxide levels
- glutathione: oxidation and reduction
   - glutathione (GSH): sulfhydryl component that serves as a general reducing agent
   - glutathione peroxidase: generates oxidized glutathione (GS-SG) during peroxide reduction
                                                                       Biochemistry: NOTES & OBJECTIVES (page 74 of 165)



   - glutathione reductase: reduction of GS-SG, regeneration of GSH
      - reaction:              GS-SG  2 GS-H [NADPH + H+  NADP+]
      - enzyme:                glutathione reductase
      - distribution:          near glutathione peroxidase
                               - NADPH produced from oxidative reactions of pentose phosphate pathway
                               - importance of glutathione reductase underscores need for functional PP pathway
reactive oxygen species can damage most macromolecules
- nucleic acids: ●OH
   - DNA damage near the site of generation, due to short hydroxyl radical half life
      - sugar: causes strand breakage
      - base: causes base modification (alteration, oxidation) or elimination
   - prevalence: 104-105 DNA base modifications per cell per day, all of which must be repaired
- proteins: O2-, HOOH, ●OH
   - O2-:        inactivation of Fe-S centers in metabolic enzymes
   - HOOH: oxidation of sulfhydryl groups
   - ●OH:        abstraction of a H atom (esp. histidine, proline, arginine, lysine)
- lipids: ●OH
   - hydroxyl radical causes abstraction of H atom, and extensive peroxidation occurs
   - this decreases membrane fluidity by affecting packing of fatty acid chains
repair of damage
- lipid peroxidation: glutathione peroxidase
   - glutathione peroxidase specificity
      - highly specific for glutathione
      - relatively nonspecific for peroxide substrate
   - function: “repair” of biomolecules converted to hydroperoxides by oxidation
      - prevention: detoxification of HOOH
      - repair: detoxification of ROOH, giving less damaging alcohols (particularly important in lipids)
   - reactions
      - hydrogen peroxide: 2 GSH + HOOH  GS-SG + 2 HOH
      - other peroxides:       2 GSH + ROOH  GS-SG + ROH + HOH
- oxidized protein: glutathione peroxidase
   - reaction: 2 GS-H + enz-S-S-R  GS-SG + RSH + enz-SH
   - function: restore sulfhydryls in oxidized proteins
   - some proteins will not be repaired, and instead will be selectively degraded by proteosomes
- damaged DNA: host excision repair
clinical relevance of oxidative stress
- red blood cells and reactive oxygen species
   - importance of ROS metabolism in RBCs
      - relatively large numbers of superoxide radicals formed from spontaneous Fe 2+ oxidation
      - gives elevated HOOH (superoxide dismutase) and ●OH (reaction with Fe2+)
         - HOOH: oxidation of sulfhydryls of proteins
         - ●OH: lipid peroxidation, weakening of RBC membrane
      - thus it is important to have this under control
   - non-enzymatic molecules in ROS metabolism
      - GSH: used to repair lipid peroxidation, disulfide bonds, as well as eliminate hydrogen peroxids
      - NADPH: pentose phosphate product that reduces GSH
      - NADH: used by methemoglobin reductase to reduce methemoglobin iron, make it functional
   - defects or deficiencies in glucose 6-phosphate dehydrogenase
      - unable to maintain cellular NADPH, which slows glutathione reductase reaction
      - peroxides will not be detoxified, and accumulating HOOH and O 2- will give rise to ●OH
      - ultimately, this can cause lipid peroxidation and cell lysis (hemolyzation)
- ROS increase in response to environmental factors
   - possible factors
      - ionizing radiation
      - redox-active drugs
      - infection
                                                                        Biochemistry: NOTES & OBJECTIVES (page 75 of 165)



   - respiratory burst oxidase (NADPH oxidase): response to infection
      - reaction: 2 O2  2 O2- + H+ [NADPH  NADP+]
      - enzyme: NADPH oxidase
         - enzyme is found in inactive form in phagocytes, B lymphocytes
         - during infection, enzyme is functionally assembled in plasma membrane
      - superoxide and secondary products (HOOH, HOCl, ●OH) work to kill invaders
      - myeloperoxidase: H2O2 with Cl- to produce HOCl
      - Chronic Granulomatous Disease: defect in an enzyme causing difficulty in defending against microbes
- Amyotrophic Lateral Sclerosis (ALS): Lou Gehrig’s disease
   - symptoms: death of motor neurons in brain and spinal cord; fatal within 5 years of onset
   - cause: unknown
      - some data suggests defects in cytoplasmic Cu/Zn superoxide dismutase may be partially responsible
      - 10 % of ALS cases are familial; of these, 20% have mutations in Cu/Zn SOD
- reperfusion injury
   - reperfusion: reoxygenation of a previously ischemic/hypoxic tussue
      - example: reoxygenation of tissue lacking, usually following constriction of blood flow
      - clinical importance: organ preservation, myocardial infarction, cerebral strokes
   - reactive species may be partially responsible
      - ischemia
         - decline in oxidative phosphorylation, ATP reduction, and increase in ADP and AMP
         - hypoxanthine accumulates due to increase in purine degradation
      - reperfusion
         - some of the increased hypoxanthine, xanthine will incompletely reduce oxygen via xanthine oxidase
         - this gives superoxide anions, which can injure tissue
- defects in oxidative phosphorylation
   - mitochondrial genome: 37 genes
      - 13 encode proteins required for oxidative phosphorylation
      - the rest encode tRNAs or rRNAs required for translation
      - several mitochondrial diseases attributed to mutations in mitochondrial genome
   - several diseases attributed to defects in ETC/OX-PHOS
      - ETC/OX-PHOS: electron transport chain and oxidative phosphorylation
      - some pathology is due to reactive oxygen species
      - defects cause buildup of reduced components of ETS (esp. CoQ), which increases formation of ROS




36. Overview – Feed/Fast Cycle & Blood Glucose Homeostasis;
       Regulation by Insulin and Glucagon
Study Guide
know the following:
- fed state
   - insulin: released from pancreatic β cells in response to high blood glucose
      - GLUT-2: high Km glucose transporter
      - glucokinase: kigh Km phosphorylation of glucose (generating glucose 6-phosphate)
      - stimulates glucose utilization, storage in liver, muscle, and adipose tissues
   - insulin-independent tissues (brain, RBCs, renal medulla): require constant supply of glucose
- post-absorptive, fasting states
   - insulin: blood glucose gradually drops to the low side of normal, causing a decrease in insulin release
   - glucagon: released from the pancreatic α cells in response to low blood glucose
      - stimulates glycogenolysis and gluconeogenesis in liver, and fatty acid release from adipose tissue
      - liver: exports glucose to the blood in order to meet the needs of the brain and RBCs
   - I/G ratio: controlling factor in rate of use, export in liver and other tissues
- glycogen storage
                                                                        Biochemistry: NOTES & OBJECTIVES (page 76 of 165)



  - liver store is major store in body, but still only lasts half a day during a fast
  - gluconeogenesis
     - ATP-requiring process, with energy coming from fatty acid fuel
     - during prolonged fast, most blood glucose comes from gluconeogenesis (especially using muscle protein)
  - glucose use regulated by hormonal control of cAMP levels, subsequent cAMP-dependent phosphorylations


Notes: Lecture and Reading
- contrasting metabolic states
   - fed state: exogenous nutrients are used, energy is stored
   - fasting state: stored nutrients are utilized to provide energy necessary for vital functions
   - insulin: pancreatic hormone signaling high blood [glucose]; higher in fed state
   - glucagon: pancreatic hormone signaling low blood [glucose]; higher in fasted state
blood glucose availability regulates the grand scheme of human energy metabolism
- insulin
   - origin:         pancreatic β cells of the endocrine pancreas
   - function:       promote blood glucose utilization, nutrient storage
   - targets:        - increases glucose uptake in muscle, adipose tissue
                     - promotes glycogen storage in muscle, liver
                     - promotes glycolysis in liver, adipose tissue, and then fatty acid synthesis from acetyl-CoA
                     - promotes protein synthesis from dietary amino acids in muscle, other tissues
   - regulation: released in response to increased blood [glucose]
                     - pancreatic β cells contain a high Km GLUT-2 glucose transporter
                     - with elevated blood [glucose], more glucose enters cell
                     - high Km glucokinase phosphorylates glucose, starts it in glycolysis
                     - transient increase in cellular ATP initiates a signal series that causes release of insulin
- glucagon
   - origin:         pancreatic α cells of the endocrine pancreas
   - function:       promote blood glucose production
   - regulation: released in response to decreased blood [glucose] (“glucose gone”)
- insulin/glugagon (I/G) ratio
   - after a carbohydrate-rich meal:
      - blood [glucose] increases       ~ 80 mg/dl to 140 mg/dl
      - insulin increases               ~ 10 μU/mL to 140 μU/mL
      - glucagon falls off              ~125 μμg/mL to 90 μμg/mL
   - the state of metabolism is determined by the I/G ratio
maintenance of blood glucose is essential
- tissue requirements
   - brain and RBCs must have glucose, as they are not able to utilize other forms of energy
   - brain:      120 g glucose / day
   - RBCs: 40 g glucose / day
   - total:      160 g / day
- glycogen stores
   - liver:      75 g glycogen available for export to the blood (lean, 70 kg human)
   - liver glycogen cannot satisfy the needs of the brain and blood for more than half a day
- producing glucose and maintaining energy
   - gluconeogenesis: de novo synthesis of glucose, with carbon derived from lactate, glycerol, and amino acids
      - glycerol: released from triglyceride in adipose tissue
      - amino acids: released from protein breakdown, primarily from muscle
   - fasting state: glucagon
      - stimulates glycogenolysis, gluconeogenesis, and export of glucose to blood
      - stimulates lipolysis in adipose tissue, with resultant release of fatty acids into the blood
         - fatty acids are used as an energy source by many tissues in lieu of glucose
         - fatty acids are also used to make ketone bodies (β-hydroxylbutyrate, acetoacetate), another energy source
         - glucose is thus saved for use by the brain, RBCs
   - some fatty acid fuel made available to liver is used to make ATP to drive gluconeogenesis
                                                                            Biochemistry: NOTES & OBJECTIVES (page 77 of 165)



maintenance of blood glucose during fasting
- blood glucose concentration units
   - clinical preference:      mg/dL (mg/100 mL, or mg %)
   - scientific preference: mM
   - MWglucose:                180 g/mol
                                    mg        g          g            mol
   - example:                   180      (1.8 ) / (180     )  0.010      10 mM
                                     dl       L         mol            L
   - reference:                - 180 mg/dL = 10 mM
                               - 90 mg/dL = 5 mM
- fasting: first 3 days
   - glucose:                  drops to near minimum (60-70 mg/dL), stays there for duration of fast
   - insulin:                  gradually decreases to minimum by 3 days of fasting
   - glucagon:                 reaches maximum at 3 days of fasting
   - I/G ratio:                lowest at 3 days of fasting
   - fatty acids:              reach maximum levels by 3 days of fasting
   - ketone bodies:            increase over duration of fast
- fasting: day 4 – week 6
   - overview
      - brain:                 adapts, begins using ketone bodies as a fuel in lieu of glucose
      - muscle:                stops using ketone bodies so that they are used almost exclusively in the brain
      - gluconeogenesis:       gradually declines
      - net result: less glucose used by the brain, so less amino acid required as a gluconeogenic substrate
         - during starvation, death is ultimately caused by excessive loss of muscle protein
         - with this, less protein needs to be broken down in muscle to provide the amino acid substrate
   - TABLE: Five Stages of Glucose Homeostasis
                      I                    II                   III                  IV                   V
      origin of       exogenous            glycogen, hepatic    glycogen, hepatic    hepatic and renal    hepatic and renal
      blood glucose                        gluconeogenesis      gluconeogenesis      gluconeogenesis      gluconeogenesis
      changes in      use by all tissues   muscle and adipose   muscle and adipose   brain, RBCs, renal   brain (diminished),
      glucose use                          tissue: diminished   tissue: gradual      medulla only         RBCs, renal medulla
                                           rates                cessation
      major fuel of   glucose              glucose              glucose              glucose, ketone      glucose (less),
      brain                                                                          bodies               ketone bodies


cAMP-mediated regulation of hepatic glucose utilization and output
- adrenalin: increased cAMP
   - binds β-adrenergic receptor, which activates adenylate cyclase via Gs heterotrimeric αβγ
   - increased cAMP activates protein kinase A, which activates target enzymes
- glucagon: increased cAMP (nearly identical to adrenalin)
   - binds glucagon receptor, which activates adenylate cyclase via Gs heterotrimeric αβγ
   - increased cAMP activates protein kinase A, which activates target enzymes
- insulin: decreased cAMP
   - binds insulin receptor, which inhibits adenylate cyclase via GI heterotrimeric αβγ
   - insulin receptor also activates cAMP phosphodiesterase
   - net effect causes a decline in cAMP, increase in 5’ AMP, and less activation of protein kinase A




37. Gluconeogenesis
Study Guide
know the following:
- gluconeogenesis: the process of synthesis of glucose from substrates such as lactate, amino acids, and glycerol
- gluconeogenesis from pyruvate: four new reactions to bypass the three irreversible steps (ATP required)
   - reactions
                                                                       Biochemistry: NOTES & OBJECTIVES (page 78 of 165)



      - pyruvate kinase
         - pyruvate carboxylase:                  pyruvate  oxaloacetate [biotin: H2CO3 ; ATP  ADP + Pi]
         - malate dehydrogenase:                  oxaloacetate  malate [NADH  NAD+]
         - (malate shuttle):                      malatemitochondria  malatecytoplasm
         - malate dehydrogenase:                  malate  oxaloacetate [NAD+  NADH]
         - PEP carboxykinase:                     oxaloacetate  phosphoenolpyruvate [GTP  GDP;  CO2]
      - phosphofructokinase-1
         - fructose 1,6-bisphosphatase-1:         fructose 1,6-bisphosphatase  fructose 6-phosphate [H2O  Pi]
      - hexokinase / glucokinase
         - glucose 6-phosphatase:                 glucose 6-phosphate  glucose [H2O  Pi]
   - regulation
      - pyruvate kinase (liver): cAMP-dependent phosphorylation of protein kinase A
         - pyruvate kinase-P:           inactive           low insulin, high glucagon        slows glycolysis
         - pyruvate kinase:             active             high insulin, low glucagon        hastens glycolysis
      - phosphoenolpyruvate carboxykinase (PEPCK)
         - high insulin:       transcriptional inhibition
         - high glucagon:      transcriptional activation
      - phosphofructokinase-1 / fructose bisphosphatase-1
         - FBPase: active with low fructose 2,6-bisphosphate (due to active FBPase-2)
         - PFK-1: active with high fructose 2,6-bisphosphate (due to active PFK-2)
         - FBPase-2 / PFK-2: regulation by protein kinase A and constitutive phosphoprotein phosphatase
            - high insulin: low protein kinase A, active PFK-2, inactive FBPase-2
            - high glucagon: high protein kinase A, active FBPase-2, inactive PFK-2
- gluconeogenesis from glycerol: two reactions to generate dihydroxyacetone phosphate
   - glycerokinase:                               glycerol  glycerol 3-phosphate [ATP  ADP]
   - glycerol 3-phosphate dehydrogenase: glycerol 3-P  dihydroxyacetone-P [NAD+  NADH + H+]
- gluconeogenesis from amino acids: converted to TCA intermediates, which are converted to pyruvate
   - alanine:         alanine  pyruvate                   [alanine aminotransferase: α-KG  Glu]
   - aspartate:       aspartate  oxaloacetate             [aspartate aminotransferase: α-KG  Glu]
   - glutamate:       glutamate  α-ketoglutarate          [glutamate DH: H2O  NH3; NAD+  NADH + H+]

Notes: Lecture and Reading
- gluconeogenesis: overview
   - gluconeogenesis: process of making glucose from non-sugar materials
      - uses many enzymes of glycolysis, but with bypass pathways around the irreversible reactions
      - phosphate bond energy used to make these bypass reactions also irreversible
   - considerations
      - localization
         - early fasting: liver, some kidney
         - prolonged fasting: primarily kidney
      - fatty acid oxidation: generates NTPs used during gluconeogenesis
      - amino acid catabolism: generates sources of carbon for gluconeogenesis
phosphates are required for conversion of fructose 1,6-bisphosphate to glucose
- forward pathway (glycolytic)
   - glucokinase:                       glucose  glucose 6-P [ATP  ADP]
   - phosphoglucose isomerase:          glucose 6-P  fructose 6-P
   - phosphofructokinase-1:             fructose 6-P  fructose 1,6-bisP [ATP  ADP]
- reverse pathway (gluconeogenic)
   - fructose 1,6-bisphosphatase-1: fructose 1,6-bisP  fructose 6-P [H2O  Pi]
   - phosphoglucose isomerase:          frucose 6-P  gluctose 6-P
   - glucose 6-phosphatase:             glucose 6-P  glucose [H2O  Pi]
- new enzymes
   - fructose 1,6-bisphosphatase-1: bypasses the second irreversible reaction of glycolysis
   - glucose 6-phosphatase: bypasses the first irreversible reaction of glycolysis
- genetic deficiencies in glucose 6-phosphatase
   - frequency: 1/200,000
                                                                      Biochemistry: NOTES & OBJECTIVES (page 79 of 165)



   - symptoms: hypoglycemia during fasting, glycogen accumulation in liver (hepatomegaly)
gluconeogenesis from pyruvate
- forward pathway (glycolytic)
   - pyruvate kinase:            phosphoenolpyruvate  pyruvate [ADP  ATP]
- reverse pathway (gluconeogenic)
   - pyruvate carboxylase:                pyruvate + carbonic acid  oxaloacetate [biotin: ATP  ADP + Pi]
   - malate dehydrogenase:                oxaloacetate  malate [NADH  NAD+]
   - (malate shuttle):                    malatemitochondria  malatecytoplasm
   - malate dehydrogenase:                malate  oxaloacetate [NAD+  NADH]
   - PEP carboxykinase:                   oxaloacetate  phosphoenolpyruvate [GTP  GDP;  CO2]
- mitochondrial transport
   - oxaloacetate generated by pyruvate carboxylase must be exported from the mitochondria
   - however, it must be converted to malate, as oxaloacetate is unable to be transported across the membrane
   - net effect
      - export of pyruvate via formation of phosphoenolpyruvate (and glycolytic bypass)
      - export of reducing equivalents (to supply glyceraldehyde 3-P DH reaction of gluconeogenesis)
biotin as a vitamin
- biotin
   - location: bound as an amide to the ε-amino of a lysyl residue on the enzyme
   - function: carboxylations (addition of CO2, from H2CO3, to an organic linkage)
   - reaction: - CO2 forms an unstable covalent intermediate with biotin
                 - it is subsequently transferred to the substrate
- deficiencies
   - biotin: cofactor vitamin needed in very tiny amounts
      - covalently bound to enzymes, so does not diffuse out of cells
      - deficiencies therefore not usually produced by dietary shortages
   - avidin: protein in raw egg white that tightly binds, complexes biotin
      - this can produce a biotin deficiency
      - cooking: renders avidin ineffective
gluconeogenesis from glycerol
- pathway
   - glycerokinase:                       glycerol  glycerol 3-P [ATP  ADP]
                                          - energetics: irreversible
                                          - localization: cytoplasm
   - glycerol 3-P dehydrogenase: glycerol 3-P  dihydroxyacetone phosphate [NAD +  NADH + H+]
                                          - energetics: reversible
                                          - localization: cytoplasm
- reactions are also used in triglyceride synthesis
gluconeogenesis from amino acids
- amino acids and gluconeogenesis
   - each α-amino acid corresponds in structure to an α-keto acid of the TCA cycle
   - thus one can make TCA intermediates by replacing the amino acid α-amino groups with α-keto groups
- example reactions
   - alanine (C3)  pyruvate
   - aspartate (C4)  oxaloacetate
   - glutamate (C5)  α-ketoglutarate
regulation of gluconeogenesis
- energy requirements for gluconeogenesis
   - during gluconeogenesis, nutritional requirements of liver must be met before glucose can be exported
   - this is done primarily by fatty acid oxidation, producing high ATP, NADH, and acetyl-CoA
   - this will allosterically slow glucose oxidation and promote gluconeogenesis
- regulation: major controls
   - regulation occurs at the three irreversible steps of glycolysis
      - glucagon: - promotes glucose output from the liver (glycogenolysis, gluconeogenesis)
                       - inhibits glucose utilization in the liver (glycogenesis, glycolysis)
      - insulin:       - inhibits glucose output from the liver (glycogenolysis, gluconeogenesis)
                                                                           Biochemistry: NOTES & OBJECTIVES (page 80 of 165)



                     - promotes glucose utilization in the liver (glycogenesis, glycolysis)
   - regulation focuses on preventing futile cycles from occuring
      - futile cycle: cycle of reactions that results in hydrolysis of ATP without doing useful work
      - there are three possible cycles corresponding to the three irreversible glycolytic reactions
- phosphoenolpyruvate / pyruvate cycle
   - pyruvate kinase:                   phosphorylated and inactivated by protein kinase A (high glucagon)
   - pyruvate carboxylase:              allosterically activated by acetyl-CoA
   - pyruvate dehydrogenase:            causes inhibition of acetyl-CoA (activates a kinase, producing inactive PDH-P)
   - PEP carboxykinase:                 transcriptionally regulated by glucagon (activated) and insulin (inhibited)
- fructose 1,6-bisP / fructose 6-P cycle
   - first level: regulation by ATP
      - phosphofructokinase-1:                    glycolysis                  allosterically activated by ATP
      - fructose 1,6-bisphosphatase-1:            gluconeogenesis             allosterically inhibited by ATP
   - second level: reciprocal regulation by fructose 2,6-bisphosphate
      - fructose 2,6-bisphosphate: regulatory molecule not otherwise involved in metabolism
      - cycle regulatory reactions
         - phosphofructokinase-1:                 glycolysis                  allosterically activated by fructose 2,6-bisP
         - fructose 1,6-bisphosphatase-1:         gluconeogenesis             allosterically inhibited by fructose 2,6-bisP
      - fructose 2,6-bisphosphate: production and degradation
         - phosphofructokinase-2:                  fructose 2,6-bisP         activated by phosphoprotein phosphatase
                                                                              inactivated by protein kinase A
         - fructose 1,6-bisphosphatase-2:          fructose 6-P              activated by protein kinase A
                                                                              inactivated by phosphoprotein phosphatase
      - fructose 2,6-bisphosphate: regulatory reactions
         - phosphoprotein phosphatase:            PFK-2-P (inactive)  PFK-2 (active) [H2O  Pi]
                                                  FBPase-2-P (active)  FBPase-2 (inactive) [H2O  Pi]
         - protein kinase A:                      PFK-2 (active)  PFK-2-P (inactive) [ATP  ADP]
                                                  FBPase-2 (inactive)  FBPase-2-P (active) [ATP  ADP]
   - third level: hormonal regulation of phosphofructokinase-2 and fructose 2,6-bisphosphatase-2
      - PFK-2 / FBPase-2
         - enzymes are part of a single, bifunctional protein, with activity dependent on phosphorylation
         - phosphorylation (protein kinase A) inactivates PFK-2, activates FBPase-2
         - dephosphorylation (phosphoprotein phosphatase) activates PFK-2-P, inactivates FBPase-2-P
      - hormonal regulation
         - glucagon: elevated cAMP, active protein kinase A, which promotes phosphorylation of PFK-2 / FBPase-2
         - insulin: depressed cAMP, inactive protein kinase A, and PFK-2 / FBPase-2 in dephosphorylated form
   - summary: effects of glucagon
      - glucagon elevates cAMP levels, leading to active protein kinase A
      - protein kinase A phosphorylates complex, activating FBPase-2 and inactivating PFK-2
         - FBPase-2 dephosphorylates fructose 2,6-bisphosphate, causing decreased [fructose 2,6-bisP]
         - PFK-2 is inactive, does not synthesize fructose 2,6-bisphosphate, causing decreased [fructose 2,6-bisP]
      - decreased [fructose 2,6-bisP] leads to:
         - increased gluconeogenesis (through more active FBPase-1)
         - decreased glycolysis (through less active PFK-1)
      - increased gluconeogenesis increases blood [glucose]
- glucose 6-phosphate / glucose cycle
   - glucokinase:                       allosterically inhibited by fructose 6-phosphate
                                        transcriptionally activated by insulin
   - glucose 6-phosphatase:             transcriptionally activated by glucagon
   - note: glucose 6-phosphatase is located on inside of ER, so glucose 6-P must be transported there via a carrier

TABLE: Enzyme nomenclature
  classical                                      modern                                 abbreviation
  fructose 6-P 1-kinase                          phosphofructokinase-1                  PFK-1
  fructose 6-P 2-kinase                          phosphofructokinase-2                  PFK-2
  fructose 1,6-bisP 1-phosphatase                fructose bisphosphatase-1              FBPase-1
                                                                      Biochemistry: NOTES & OBJECTIVES (page 81 of 165)



   fructose 1,6-bisP 2-phosphatase            fructose bisphosphatase-2            FBPase-2
   glucose 6-P phosphatase                    glucose 6-phosphatase                G6Pase
   phosphoenolpyruvate carboxykinase          PEP carboxykinase                    PEPCK




38., 39. Triglyceride Mobilization and Fatty Acid Oxidation
Study Guide
know the following:
- triglycerides
   - major storage form of metabolic energy in humans
      - highly reduced
      - able to be stored in a relatively anhydrous state
   - most triglyceride stored in adipose tissues
- fatty acid catabolism
   - controlled at hormone-sensitive lipase, the first step of fatty acid lipolysis
      - physiological conditions:       fasting, prolonged exercise
      - hormonal conditions:            high glucagon or high adrenaline
      - enzyme conditions:              hormone-sensitive lipase-P (active form)
   - products of lipolysis: fatty acids, glycerol
- fatty acid utilization
   - activation: addition of CoA using ATP  AMP
   - transport into mitochondria: carnitine cycle
      - FA-CoAcytoplasm  HS-CoAcytoplasm [carnitine  O-acyl-carnitine]
      - O-acyl-carnitine  carnitine [HS-CoAmitochondria  FA-CoAmitochondria]
      - enzyme: CoA:carnitine acyl transferase (CCAT)
         - inhibited by malonyl-CoA
         - malonyl-CoA produced in fatty acid synthesis with fed state, high insulin
   - β-oxidation of fatty acyl-CoA derivatives
      - step 1:      oxidation of alkane to alkene at β carbon, generating trans enoyl derivative
                     enzyme: acyl-CoA dehydrogenase (succinate DH analog: [FAD  FADH2])
      - step 2:      hydrogenation of C=C bond, forming alcohol at β carbon
                     enzyme: fumarase analog
      - step 3:      oxidation of alcohol to ketone, forming β-keto acyl-CoA derivative
                     enzyme: malate DH analog [NAD+  NADH + H+]
      - step 4:      reversible cleavage of C-C bond, adding HS-CoA and splitting off acetyl-CoA
      - net result: get fatty acyl-CoA (n-2) + acetyl-CoA
- ketone bodies
   - logic
      - by lipolysis, liver takes in more fatty acids than it can use for TCA cycle
      - citrate synthase is inhibited by high FA-CoA, leading to accumulation of acetyl-CoA
      - acetyl-CoA is used in the synthesis of ketone bodies
   - process
      - 2 acetyl-CoA  acetoacetyl-CoA [ HS-CoA]
      - acetyl-CoA + acetoacetyl-CoA  HMGCoA [H2O  HS-CoA]
      - HMGCoA  acetoacetate [ acetyl-CoA]
      - acetoacetate  β-hydroxybutyrate [βOHB DH: NADH + H+  NAD+]
      - acetoacetate  acetone [ CO2]
   - the complicated process is done to ensure that acetoacetate is only made with very high acetyl-CoA
   - utilization
      - βOHB DH: β-hydroxybutyrate  acetoacetate [NAD+  NADH + H+]
      - acetoacetate + succinyl-CoA  acetoacetyl-CoA + succinate
                                                                      Biochemistry: NOTES & OBJECTIVES (page 82 of 165)



    - acetoacetyl-CoA  2 acetyl-CoA [HS-CoA ]


Notes: Lecture and Reading
- energy storage in triglycerides
   - triglycerides have more potential energy than fat stored as glycogen
      - more reduced: more efficient fuel per gram anhydrous weight
      - more anhydrous storage: less bound water required to complex triglycerides
   - localization
      - adipose tissue:        ~85 %
      - skeletal muscle:       15 %
      - liver:                 0.4 %
overview of fat catabolism
- adipose tissue
   - triglycerides broken down into glycerol, fatty acids via lipolysis
   - fatty acids are exported into blood
- liver
   - 25% of blood FA imported
   - fatty acids activated to FACoA
   - FACoA imported to mitochondria
   - FACoA converted to acetyl-CoA by β-oxidation
   - some acetyl-CoA used to sustain liver; the rest is converted to ketone bodies and exported from mitochondria
   - ketone bodies are exported from liver into bloodstream
- muscle
   - fatty acids:
      - 75% of blood FA imported
      - fatty acids activated to FACoA
      - FACoA imported to mitochondria
      - FACoA converted to acetyl-CoA by β-oxidation
      - acetyl-CoA entered into TCA cycle, converted to ATP
   - ketone bodies
      - ketone bodies imported, bought into mitochondria
      - converted to AcAcCoA, to acetyl-CoA
      - acetyl-CoA entered into TCA cycle, converted to ATP
lipolysis: mobilization of fatty acids from adipose tissue
- lipases
   - hormone-sensitive lipase:         triacylglycerol + H2O  diacylglycerol + FA (rate-limiting)
   - hormone-insensitive lipases:      diacylglycerol + H2O  monoacylglycerol + FA
                                       monoacylglycerol + H2O  glycerol + FA
- hormonal regulation
   - hormone-sensitive lipase: phosphorylated, activated by protein kinase A, which is regulated by cAMP
   - adrenaline: increases cAMP  increased FA production
   - glucagon:       increases cAMP  increased FA production
   - insulin:        decreases cAMP  decreased FA production
- fates
   - fatty acids: - released to the blood and complexed with albumin for transport
   - glycerol:       - cannot be rephosphorylated to glycerol 3-P, as adipose tissue lacks glycerol kinase
                     - must be transported to liver for gluconeogenesis
oxidation of fatty acids
- activation
   - reaction:       R-COOH  (R-COO-AMP)enz [ATP  PPi]  R-CO-S-CoA [HS-CoA  AMP]
   - mechanism: formation of high energy carboxyphosphate intermediate, followed by thioester formation
   - energetics: requires 2 ATP (PPi + H2O  2 Pi)
- transport into mitochondria
   - reaction:       FA-CoAcytoplasm + carnitine  O-acyl-carnitine + HS-CoAcytoplasm
                     O-acyl-carnitine + HSCoAmitochondria  FA-CoAmitochondria + carnitine
                                                                          Biochemistry: NOTES & OBJECTIVES (page 83 of 165)



   - enzyme:          carnitine acyl transferase (CCAT)
   - regulation: malonyl-CoA (intermediate of FA biosynthesis)
- β-oxidation of fatty acyl-CoA derivatives
   - step 1
      - mechanism:             oxidation of alkane to alkene at β carbon, generating trans enoyl derivative
      - cofactors:             [FAD  FADH2]
      - enzyme:                acyl-CoA dehydrogenase (analog of succinate dehydrogenase)
   - step 2
      - mechanism:             hydrogenation of C=C bond, forming alcohol at β carbon
      - enzyme:                (analog of fumarase)
   - step 3
      - mechanism:             oxidation of alcohol to ketone, forming β-keto acyl-CoA derivative
      - cofactors:             [NAD+  NADH + H+]
      - enzyme:                (analog of malate dehydrogenase)
   - step 4
      - mechanism:             reversible cleavage of C-C bond, adding HS-CoA and splitting off acetyl-CoA
   - net result
      - get fatty acyl-CoA (n-2) + acetyl-CoA
      - cycle is repeated until fatty acid has been completely converted to acetyl-CoA
      - at end of cycle, get 2 acetyl-CoA (for even chain fatty acids)
considerations in β-oxidation
- energy yield (shown with palmitate as an example)
   - net reaction: palmitate + 7 HS-CoA + 7 H2O + 7 NAD+  8 acetyl-CoA + 7 NADH + 7 H+ + 7 FADH2
   - reaction products
      - β-oxidation (x7):                         gives 8 acetyl-CoA + 7 NADH + 7 FADH2
      - TCA cycle, acetyl-CoA (x8):               gives 16 CO2 + 24 NADH + 8 FADH2
   - ATP yield
         8 substrate-level phosphorylations (GDP  GTP):            8 ATP
         31 NADH through oxidative phosphorylation:                 93 ATP
         15 FADH2 through oxidative phosphorylation:                30 ATP
         cost of palmitate activation:                              - 2 ATP
         total:                                                     129 ATP (net)
- control of β-oxidation
   - once inside mitochondria, only rate of FAD, NAD+ regeneration control β oxidation
   - thus rate of electron transport controlled by ADP availability (respiratory control)
- genetic deficiencies in β-oxidation
   - three types of fatty acyl-CoA dehydrogenases
      - long chain:            > C12
      - medium chain:          C6 – C12
      - short chain:           < C6
   - medium chain deficiencies common (1/10,000)
   - symptoms: severe fasting hypoglycemia, but otherwise do well if prolonged fasting is avoided
   - reason: alternate pathways of fatty acid oxidation, such as those in peroxisomes
- oxidation of odd-chain fatty acids
   - oxidized normally until C5 acyl derivative is reached
   - thiolytic cleavage with HS-CoA generates acetyl-CoA, propionyl-CoA
   - propionyl-CoA enters metabolic sequence as succinyl-CoA (through methylmalonyl-CoA)
- β-oxidation of unsaturated fatty acyl-CoA (SUPPLEMENTARY)
   - unsaturated fatty acids occur in high concentrations in human depot fat
   - two additional enzymes required for β-oxidation
      - first shifts cis double bond to trans double bond at the proper position
      - second racemizes β-hydroxy intermediate to the proper stereoisomer
formation and utilization of ketone bodies
- overview: ketone bodies
   - examples:        acetone, acetoacetate (AcAc), and β-hydroxybutyrate (βOHB)
   - synthesis:       response to extensive fasting, where fatty acids are major fuel (fasting, prolonged exercise)
                                                                      Biochemistry: NOTES & OBJECTIVES (page 84 of 165)



   - localization: liver mitochondria only
   - regulation: acetyl-CoA accumulates because β-oxidation is faster than TCA
                      - TCA allosterically slowed by NADH, citrate
                      - also allosterically slowed by high fatty acyl-CoA
- synthesis
   - 2 acetyl-CoA  acetoacetyl-CoA [ HS-CoA]
   - acetyl-CoA + acetoacetyl-CoA  β-hydroxy-β-methylglutaryl-CoA (HMGCoA) [H2O  HS-CoA]
   - HMGCoA  acetoacetate [ acetyl-CoA]
      - acetoacetate  acetone [ CO2]
      - acetoacetate  β-hydroxybutyrate [β-hydroxybutyrate dehydrogenase: NADH + H+  NAD+]
- utilization
   - process
      - AcAc and βOHB transported from liver to extrahepatic tissues (primarily skeletal muscle, heart)
      - β-hydroxybutyrate dehydrogenase: in mitochondria, reversal of last reaction; converts βOHB to AcAc
      - transferase: acetoacetate + succinyl-CoA  acetoacetyl-CoA + succinate
      - acetoacetyl-CoA cleaved to acetyl-CoA (reversal of first reaction), entered into TCA cycle
   - timing
      - during prolonged starvation, brain adapts to use of ketone bodies as metabolic fuels
      - skeletal muscle in well-conditioned athletes is more efficient at utilizing these compounds
- ketosis
   - ketosis will result in net accumulation of H+, causing blood pH to fall (palmitate  4 acetoacetate- + 4 H+)
      - ketosis: accumulation of ketone bodies in the blood
      - ketonuria: accumulation of ketone bodies in the urine
      - acidosis: lowering of blood pH
   - starvation leads to ketoacidosis, though milder than what is seen in diabetis
ketone bodies and gluconeogenesis
- ketone bodies formed under same physiological conditions as what favors gluconeogenesis
- cannot be converted in net amounts to glucose: acetyl-CoA oxidized to two CO2 in one turn of the TCA cycle
summary: key regulatory events governing fat catabolism
- stored body fat catabolized in any fast lasting more than 4-6 hours
- initiated by drop in I/G in blood, which declines to minimum after 2-3 days of fasting
- leads to activation of lipolysis and the following:
   - adipose: increased cAMP  active PKA  active hormone-sensitive lipase, increase in FA and glycerol
   - liver takes up fatty acids, transports into mitochondria
      - due to active CoA:carnitine acyl transferase (CCAT) resulting from low malonyl-CoA
      - malonyl-CoA low because FA synthesis not taking place due to low insulin
   - fatty acid oxidation keeps ATP/ADP and NADH/NAD+ ratios high in most tissues
      - rate of fatty acid oxidation limited only by rate of electron transport
      - this is governed by availability of NAD + and ADP
   - citrate synthase: inhibited in liver by FACoA, NADH, resulting in acetyl-CoA accumulation in mitochondria
   - increase in mitochondrial acetyl-CoA gives ketone body synthesis by mass action




40., 41. De Novo Synthesis of Fatty Acids from Carbons of Glucose
Study Guide
know the following:
- fatty acid synthesis: overview
   - location:               liver, adipose
   - signal:                 plentiful carbohydrate (high insulin)
   - carbon source:          glucose
   - requirements:           ATP (glucose catabolism)
                             NADPH (pentose-P, citrate shuttle)
                                                                     Biochemistry: NOTES & OBJECTIVES (page 85 of 165)



                                acetyl-CoA in the cytoplasm (citrate shuttle)
- fatty acid synthesis: process
   - citrate shuttle
      - citrate synthase:               acetyl-CoA + oxaloacetate  citrate [H2O  HS-CoA]
      - (carrier protein):              citratemitochondria  citratecytoplasm
      - ATP citrate lyase:              citrate  oxaloacetate + acetyl-CoA [HS-CoA , ATP  ADP + Pi]
      - malate DH:                      oxaloacetate  malate [NADH + H+  NAD+]
                +
      - NADP -dep. malate DH:           malate  pyruvate [NADP+  NADPH;  CO2]
      - (carrier protein):              pyruvatecytoplasm  pyruvatemitochondria
      - pyruvate carboxylase:           pyruvate  oxaloacetate [biotin: H2CO3 , ATP  ADP + Pi]
   - pentose phosphate pathway
      - glucose 6-P DH:                 glucose 6-P  6-phosphogluconolactone [NADP+  NADPH + H+]
      - (some enzyme)                   6-phosphogluconolactone  6-phosphogluconate [H2O ]
      - 6-phosphogluconate DH:          6-phosphogluconate  ribulose 5-P [NADP+  NADPH + H+;  CO2]
   - fatty acid synthesis
      - acetyl-CoA carboxylase:         acetyl-CoA  malonyl-CoA [biotin: H2CO3 ; ATP  ADP + Pi]
      - fatty acid synthase:            acetyl-CoA (or elongating FA) transferred to HS-ACP ( HS-CoA)
                                        malonyl-CoA transferred to HS-synthase ( HS-CoA)
                                        elongating FA condensed onto malonyl-CoA ( CO2)
                                        β-keto group reduced in three steps (reversal of β-oxidation):
                                        - β-keto group reduced to hydroxyl group [NADPH + H +  NADP+]
                                        - double bond formed [ H2O]
                                        - double bond reduced [FADH2  FAD  FADH2 (NADPH + H+  NADP+)]
                                        carbon fragment transferred to HS-ACP
      - 8 acetyl-CoA + 14 (NADPH + H+) + 7 ATP + H2O  palmitic acid + 8 HS-CoA + 14 NADP+ + 7 (ADP + Pi)
- triglyceride synthesis
   - glycerokinase (liver only):        glycerol  glycerol 3-P [ATP  ADP]
   - glycerol 3-phosphate DH:           dihydroxyacetone-P  glycerol 3-P [NADH + H+  NAD+]
   - (activation):                      3 FA  3 FA-CoA [3 CoASH ; 3 (ATP  AMP + 2 Pi)]
   - (esterification):                  2 FA-CoA + G3P  L-phosphatidic acid (diglyceride-P) [ 2 CoASH]
   - (hydrolysis):                      L-phosphatidic acid  diglyceride [H2O  Pi]
   - (esterification):                  diglyceride + FA-CoA  triglyceride [ CoASH]
- triglyceride transport: attached to VLDL, transported to target tissue through bloodstream
- triglyceride unloading
   - hydrolysis by lipoprotein lipase, forming free FA and glycerol
      - glycerol: transported back to liver
      - FA: taken in by cells, resynthesized as triglycerides
   - with enough triglyceride removal, VLDL  LDL
- regulation: fatty acid biosynthesis
   - acetyl-CoA carboxylase
      - insulin
         - allosteric: insulin-dependent kinase; phosphorylation and inactivation
         - adaptive: IRE induces synthesis of acetyl-CoA carboxylase and other lipogenic enzymes
      - glucagon
         - allosteric: cAMP activates PKA; phosphorylation and inactivation at different site
      - polyunsaturated fatty acids:
         - allosteric: polyunsaturated fats suppress induction of lipogenic enzymes
   - malonyl-CoA: accumulates when acetyl-CoA carboxylase is active
      - function: inhibits CoA:carnitine acyl transferase
      - logic: prevents oxidation of newly-synthesized fatty acyl-CoA, indirectly pushes towards FA esterification
   - lipoprotein lipase: activated by insulin
   - GLUT-4 transporter: activated by insulin


Notes: Lecture and Reading
overview of fat synthesis and storage in the fed state
                                                                   Biochemistry: NOTES & OBJECTIVES (page 86 of 165)



- de novo fatty acid biosynthesis
   - locations
      - liver: major site
      - adipose tissue: minor site
   - liver
      - glucose intake
         - after a meal, concentration of glucose and insulin will be highest in the portal vein
         - portal vein: flow from the gut to the liver (strategic for metabolism)
         - GLUT-2: high Km transporter used to take glucose into the liver, even at high concentrations
         - glucokinase: high Km glucose phosphorylation protein
         - glucose that is not taken in is allowed to flow through the rest of the body
      - fatty acid synthesis
         - glucose is converted to glycogen, also metabolized in glycolysis to pyruvate
         - pyruvate is converted to acetyl-CoA, some of which enters TCA and some of which enters FA synthesis
            - requires NADPH for biosynthetic reactions
            - NADPH derived partially from pentose phosphate pathway
         - fatty acids esterified to triglycerides
      - transport: triglycerides attached to VLDL and put into circulation
   - adipose tissue
      - storage: triglyceride is removed from VLDL, stored in the tissue
      - synthesis: triglyceride can be made from the same reactions as used in the liver
- dietary absorption
   - absorption: intestinal mucosa cells
   - transport: packaged on lipoprotein particles called chylomicrons, transported to blood via lymph
   - storage: removed from chylomicrons at adipose tissue, stored there
DE NOVO FATTY ACID SYNTHESIS AND TRIGLYCERIDE STORAGE
de novo synthesis of fatty acids
- overview: palmitic acid
   - synthesis: cytoplasm
      - 8 acetyl-CoA + 14 (NADPH + H+) + 7 ATP + H2O  palmitic acid + 8 HSCoA + 14 NADP+ + 7 ADP + 7 Pi
      - enzymes: acetyl-CoA carboxylase, fatty acid synthase
      - other enzymes then modify palmitic acid (desaturation, elongation)
   - acetyl-CoA
      - source: glycolysis (cytoplasm), pyruvate dehydrogenase (mitochondrial matrix)
      - because pyruvate DH occurs in the mitochondria, a transport system (the citrate shuttle) is necessary
   - citrate shuttle
      - each cycle
         - transfers one acetyl-CoA from mitochondrial matrix to cytoplasm
         - generates one NADPH from NADP+
      - repetitions: palmitate synthesis
         - 8 shuttles required to generate the 8 acetyl-CoA
         - 8 NADPH are supplied by these shuttles, but 6 more are required
         - other 6 NADPH come from pentose phosphate pathway
- citrate shuttle: provides cytosolic acetyl-CoA, NADPH
   - citrate synthase:                    oxaloacetate + acetyl-CoA --> citrate [H2O ,  HSCoA]
   - (carrier protein, MC):              citratemitochondria  citratecyt
   - ATP citrate lyase:                   citrate  oxaloacetate + acetyl-CoA [HSCoA , ATP  ADP + Pi]
   - malate dehydrogenase:                oxaloacetate  malate [NADH + H+  NAD+]
              +
   - NADP -dependent malate DH: malate  pyruvate + CO2 [NADP+  NADPH + H+]
   - (carrier protein, CM):              pyruvatecyt  pyruvatemitochondria
   - pyruvate carboxylase:                pyruvate + H2CO3  oxaloacetate [ATP  ADP + Pi]
   - net cost: 2 ATP
- pentose phosphate: remainder of required NADPH
   - glucose 6-P  6-phosphogluconolactone [NADP+  NADPH + H+]
   - 6-phosphogluconolactone  6-phosphogluconate [H2O ]
   - 6-phosphgluconate  ribulose 5-phosphate [ CO2, NADP+  NADPH + H+]
                                                                         Biochemistry: NOTES & OBJECTIVES (page 87 of 165)



   - net gain: 2 NADPH per glucose
- acetyl-CoA carboxylase: first committed step in fatty acid biosynthesis
   - reaction:       H2CO3 + acetyl-CoA  malonyl-CoA [biotin: ATP  ADP + Pi]
   - enzyme:         acetyl-CoA carboxylase
   - mechanism: analogous to pyruvate carboxylase
   - energetics: rate-limiting, first committed step in fatty acid biosynthesis
- fatty acid synthase
   - net reaction: acetyl-CoA + 7 malonyl-CoA + 14 (NADPH + H+) + H2O
                                palmitic acid + 14 NADP+ + 7 CO2 + 8 HSCoA + 7 H2O
   - mechanism: - malonyl acyl group of malonyl-CoA transferred to HS-ACP (acyl-carrier protein)
                     - acetyl group of acetyl-CoA transferred to another reactive sulfhydryl group on HS synthase
                     - acetyl group condenses with methylene group of malonyl-CoA, giving off CO2
                     - β-keto group reduced to methylene group by three sequential reactions (rev. of β-oxidation)
                               - ketone reduction to alcohol [NADPH + H+  NADP+]
                               - dehydration of alcohol to alkene [ H2O]
                               - alkene reduction to alkane [FADH2  FAD  FADH2 (NADPH + H+  NADP+)]
                     - elongating chain transferred to HS synthase, malonyl-CoA attaches to ACP, and cycle restarts
- modification of palmitate
   - palmitic acid: can be further elongated, desaturated by other enzymes
   - must first be activated to palmitoyl-CoA before it can be modified or incorporated into a triglyceride
synthesis of triglycerides and storage
- synthesis
   - precursor: must begin with glycerol 3-phosphate
      - glycerol 3-P dehydrogenase (several tissues): dihydroxyacetone phosphate  glycerol 3-phosphate
      - glycerokinase (liver only): glycerol  glycerol 3-phosphate
   - glycerol 3-phosphate + 2 fatty acids  L-phosphatidic acid + 2 HS-CoA
      - L-phosphatidic acid: can be used in phospholipids
   - L-phosphatidic acid  diglyceride [H2O  Pi]
   - diglyceride + fatty acid  triglyceride + CoA-SH
- transport
   - direction: liver (synthesis) to adipose (storage)
   - vehicle: very low density lipoprotein (VLDL)
      - core: hydrophobic, nonpolar molecules (cholesterol esters, triglycerides)
      - shell: cholesterol, phospholipids, proteins
- unloading
   - lipoprotein lipase
      - location: surface of endothelial cells lining the capillaries perfusing adipose tissues
      - function: hydrolyzes triglycerides, releases into capillaries
   - fatty acids: taken up by adipose tissue, resynthesized using glycerol 3-P derived from glucose
   - VLDL: converted to LDL by triglyceride removal
   - regulation: driven by high levels of insulin
regulation of fatty acid biosynthesis
- acetyl-CoA carboxylase
   - phosphorylation/dephosphorylation
      - insulin: activates an insulin-dependent kinase, phosphorylating and activating the enzyme
      - glucagon: cAMP; activates protein kinase A, which phosphorylates and inhibits the enzyme (different site)
   - adaptive changes: insulin response elements (IREs)
      - insulin: promotes increased synthesis of acetyl-CoA carboxylase, other lipogenic enzymes
      - regulation
         - carbohydrates: if continuously available, insulin is chronically high, and more fat is synthesized and stored
         - polyunsaturated fatty acids: suppress induction of these genes
      - adaptive changes require several days to be manifested due to timing of synthesis, degradation
- malonyl-CoA
   - accumulation
      - accumulates when acetyl-CoA carboxylase is active
      - in high concentration, inhibits CoA:carnitine acyl transferase
                                                                        Biochemistry: NOTES & OBJECTIVES (page 88 of 165)



   - function
      - prevents oxidation of newly-synthesized fatty acyl-CoA
      - indirectly forces fatty acyl-CoA esterification to triglycerides in the cytoplasm
- insulin
   - lipoprotein lipase: hydrolysis of triglycerides; activated by insulin
   - GLUT-4 transporter: intake of glucose for glycerol 3-P synthesis; activated by insulin
elongation of fatty acids (SUPPLEMENTARY)
- palmitate can be elongated by two systems, one mitochondrial and one in the endoplasmic reticulum
- saturated and unsaturated fatty acids can be further elongated, up to C 26
formation of mono- and polyunsaturated fatty acids (SUPPLEMENTARY)
- double bonds: introduced by an ER desaturase system that utilizes NADPH, O2
- essential fatty acids: precursors to “eicosanoids,” which are important in signalling
   - due to specificity of mamallian desaturases, not all necessary fatty acids can be made by the human body
   - limits of double bond addition do not allow one to be within 6 C of methyl terminus




42. Triglyceride Absorption, Transport, and Storage
Study Guide
know the following:
- triglyceride absorption and storage
   - intestinal mucosa
      - emulsification:      formation of mixed micelles via bile detergents to increase solubility of triglycerides
      - hydrolysis:          pancreatic lipase: hydrolyzes triglycerides into monoglycerides or glycerol, fatty acids
   - intestinal cells
      - absorption:          mucosal cells lining intestine
      - esterification:      synthesis of triglycerides from hydrolysis products (ATP-requiring process)
      - secretion:           triglycerides secreted from intestinal cells into blood
   - blood stream
      - transport:           triglycerides transported through blood on chylomicrons (CM)
      - hydrolysis:          triglycerides hydrolyzed to glycerol, fatty acids
   - target tissue
      - absorption:          free fatty acids absorbed into target tissues
      - esterification:      synthesis of triglycerides from hydrolysis products (ATP-requiring process)
- fat soluble vitamins: A, D, E, K
   - vitamins A, D, E, and K are dissolved in fat; fat must be emulsified for vitamin absorption to occur
   - vitamins incorporated into chylomicrons, typically reach liver in CMR
   - deficiencies in fat absorption: lead to deficiencies in fat-soluble vitamins, essential fatty acids


Notes: Lecture and Reading
DIGESTION AND ABSORPTION OF DIETARY FAT
- lipids: overview
   - lipids: class of biological molecules soluble in organic solvents but not water
   - examples: triglycerides, phospholipids, sphingolipids, cholesterol, steroids, fat-soluble vitamins
absorption of dietary triglycerides
- emulsification
   - purpose
      - lipid enters the diet as large insoluble droplets that are incapable of absorption by intestinal epithelium
      - emulsification increases solubility, making them better substrates for hydrolytic lipases
   - micelles: amphipathic spherical aggretates with a hydrophobic core and hydrophilic surface
   - detergent: amphipathic molecules capable of disrupting hydrophobic interactions, dispersing into mixed micelles
   - biliary secretions: synthesized in liver, stored in gall bladder, secreted into duodenum during digestion
                                                                        Biochemistry: NOTES & OBJECTIVES (page 89 of 165)



      - bile acids: wedge-shaped steroid molecules with good detergent properties; returned to liver via portal vein
      - phosphatidylcholine: component of the micelles
      - cholesterol: component of the micelles
- pancreatic lipase
   - origin:         synthesized in pancreas, secreted into duodenum
   - function:       hydrolysis of emulsified triglyceride, typically at 1, 3 positions
                     - yields a 2-acyl monoglyceride
                     - fatty acid can migrate to 1 or 3 position for complete hydrolysis, though this is not necessary
                     - approximately 60% of ingested triglyceride is as 2-acyl monoglyceride
- absorption and reesterification
   - absorption: fatty acids, monoglycerides absorbed by mucosal cells lining the intestine
   - resynthesis: intestinal mucosa resynthesized triglycerides from the hydrolysis products
                     - fatty acids must be activated to fatty-acyl CoA, an ATP-requiring process (ATP  AMP + PPi)
                     - fatty acyl-CoA derivatives used to synthesize triglycerides
                               2-acyl-monoglyceride + 2 FA-S-CoA  triglyceride + 2 HS-CoA
- secretion into the blood
   - transport in the blood is accomplished via a lipoprotein complex called a chylomicron
   - chylomicron
      - composition: “apoproteins” and other lipids such as cholesterol
      - function: serves as transport vehicle for triglyceride in the blood
      - distribution: once loaded, are secreted from mucosal cells into lymph, pass into venous blood via thoracic duct
- utilization of absorbed lipid
   - can supply to numerous tissues, depending on the nutritional state of the animal
   - process
      - lipoprotein lipase: hydrolyzes triglycerides into glycerol and fatty acids (activated by insulin)
      - fatty acids are taken in by the target, and the remaining glycerol is taken in by the liver
      - chylomicron remnant (CMR): cholesterol-rich remains of chylomicron after triglyceride removal
   - post-prandial period (following a normal meal with adequate CHO)
      - lipoprotein lipase in adipose tissue is activated, via an unknown mechanism
      - because of this, most post-prandial triglyceride is metabolized by adipose tissue
      - adipose tissue requires glucose from the blood to resynthesize triglycerides due to the lack of glycerol kinase
- fat cells sense when they are getting fat
   - leptin: adipose endocrine hormone secreted in response to excess caloric intake, weight gain
      - target: hypothalamus neuropeptides regulating appetite response (one of which is malonyl-CoA)
      - regulation: excess fat deposition, resulting increase in leptin should decrease appetite
   - clinical use: obese humans do usually make normal levels of leptin, so clinical use is limited
absorption of fat soluble vitamins and essential fatty acids
- fat soluble vitamins: A, D, E, K
   - fat soluble vitamins are literally dissolved in fat, which fat must be emulsified before absorption can occur
   - once inside, vitamins incorporated into chylomicrons for tramsport to tissues
   - most of vitamin usually reaches liver via CMR
- essential fatty acids: polyunsaturated C18, C20 acids
   - constituents of plant oil triglycerides
   - function: source of arachidonic acid (C20Δ5,8,11,14), the precursor to eicosanids
   - eicosanids: essential family of local hormones including prostaglandins, thromboxanes, leukotrienes
   - any impairment of fat absorption can cause a deficiency in fat-soluble vitamins




42., 43. Cholesterol and Lipoprotein Metabolism
Study Guide
know the following:
- cholesterol structure
                                                                         Biochemistry: NOTES & OBJECTIVES (page 90 of 165)



   - free form: unesterified (alcohol group on C3) form typically found as a membrane component
   - esterified form: form with fatty acid chain ester-linked to C3; typical storage form (Chol.E or CE)
- cholesterol sources
   - dietary intake: animal tissues, esp. eggs, dairy products, and meat
   - synthesis: de novo from acetyl-CoA precursors (usually in liver); usually forming 60% of total cholesterol
- cholesterol synthesis
   - initial steps: same as ketone body synthesis
      - 2 acetyl-CoA  acetoacetyl-CoA [ HS-CoA]
      - acetyl-CoA + acetoacetyl-CoA  HMGCoA [H2O  HS-CoA]
      - HMGCoA  acetoacetate [ acetyl-CoA]
   - HMGCoA reductase: committed step of cholesterol synthesis
      - reaction:              HMGCoA  mevalonate [2 (NADPH  NADP+);  CoA-SH]
      - mechanism:             reduction of thioester group to an alchohol, using 2 NADPH
      - energetics:            controlled, committed step of cholesterol synthesis
   - bile acids: major metabolic product of cholesterol (primary bile acids: cholic acid, chenodeoxycholic acid)
      - conjugation: carboxyl groups conjugated in amide linkage with either glycine or taurine before secretion
      - modification: conjugated acids are hydrolyzed in intestine to regenerate the unconjugated forms
- enterohepatic circulation
   - synthesis:       liver
   - storage:         gall bladder
   - circulation: secreted into intestine, absorbed by ileum, returned to liver via portal vein
   - excretion:       major route via cholesterol and bile acids that are not reabsorbed (~1 g / day)
- synthesis and secretion of bile acids
   - primary bile acids: cholic acid, chenodeoxycholic acid (synthesized in the liver from cholesterol)
   - conjugation: both primary and secondary bile acids
      - carboxyl groups conjugated in amide linkage with either glycine or taurine before release into bile duct
      - increases hydrophobic character
   - modification
      - in the intestine, conjugated acids are hydrolyzed to regenerate the free carboxyl group, glycine, and taurine
      - secondary bile acids: some primary bile acids modified by bacteria, resulting in loss of 7-hydroxy group
- enterohepatic circulation: primary means of getting rid of cholesterol
   - bile: major components and molar ratio
      - bile acids:                               16
      - phosphatidylcholine (“lecithin”):         3
      - cholesterol:                              1
   - components are synthesized in the liver, stored in the gall bladder
   - cholesterol and bile acids
      - cholesterol is highly insoluble
      - requires bile acids to emulsify the cholesterol and form mixed micelles with lecithin
      - cholestasis: cholesterol proportion too high, resulting in insoluble gall stones and obstruction of the bile duct
   - circulation
      - circulated through intestine, absorbed by ileum, returned to liver via portal vein
      - 15-30 g of circulation (2-4 g pool of bile acids, which circulates 5-10 times daily)
   - reabsorption: both primary and secondary bile acids
   - excretion
      - bile acids: 0.2-0.6 g/day pass into feces, representing the major means of cholesterol excretion
      - cholesterol: 1 g secreted into intestine; only about 0.5 g reabsorbed
      - net steroid secretion: bile acids + cholesterol = approximately 1 g
- other components of the bile
   - bilirubin: breakdown product of heme catabolism; transported to liver via albumin forexcretion
   - biliary obstruction: can lead to accumulation of bilirubin in the liver, ultimately in the blood
   - jaundice: accumulation of bilirubin or bilirubin diglucouronide in the blood, causing yellow discoloration of skin
- dietary absorption
   - intestinal mucosa
      - emulsification:        formation of mixed micelles from dietary cholesterol esters, free cholesterol
      - hydrolysis:            cholesterol esterase of emulsified cholesterols, forming free cholesterol
                                                                        Biochemistry: NOTES & OBJECTIVES (page 91 of 165)



  - intestinal cells
     - absorption:         mucosal cells of small intestine (free cholesterol only)
     - esterification:     limited amounts of free cholesterol esterified
  - bloodstream
     - transport:          free cholesterol, CE incorporated into chylomicrons
                           triglycerides dumped into target tissues, forming CMR
   - liver
      - endocytosis:           CMR receptor-mediated endocytosis absorbs CMR
      - metabolism:            cholesterol esters hydrolyzed, free cholesterol used in biosynthesis or storage
- homeostasis: input (diet, de novo) must be balanced against output (bile)
   - transcriptional control: HMGCoA reductase and other lipogenic enzymes
      - high cholesterol:      binds, inactivates steroid regulatory element (SRE) and binding protein (SREBP)
      - high insulin:          binds, activates SREBP, increasing HMGCoA reductase and other lipogenic enzymes
   - dietary control
      - high cholesterol:      decreased HMGCoA reductase activity (sensed by CMR, LDL taken in by liver)
      - low cholesterol:       increased HMGCoA reductase activity
      - caloric intake:        increases insulin, circulating FA, which promote hepatic VLDL synthesis
      - fatty acid ration:     increased polyunsaturated inhibits SRE-mediated events, causing cholesterol decrease
- cholesterol regulation: central role of liver
   - regulates synthesis inversely with dietary cholesterol, caloric intake
   - regulates excretion through synthesis of bile components (cholesterol, bile acids)
   - regulates plasma lipoprotein (VLDL, nascent HDL) synthesis
   - regulates uptake, catabolism of LDL and HDL
- liver transport to extrahepatic tissues
   - VLDL catabolism: forms LDL, which can be taken in by numerous tissues
   - classic LDL receptor-mediated endocytosis
      - binding:               LDL binds high affinity receptor in clatharin-coated plasma membrane pits
      - endocytosis:           LDL is internalized
      - maturation:            receptor returned to surface; endosome merges with lysosome, lipoprotiens degraded
      - metabolism:            biosynthesis: cell membranes, synthetic reactions
                               storage: esterification via ACAT, storage in lipid droplets
      - regulation:            HMGCoA reductase: feedback inhibition by cholesterol internalized by this mechanism
                               LDL receptors: reduced in number by cholesterol feedback
                               ACAT: activated by excess cholesterol
   - scavenger cell pathway: not well characterized; known to utilize another type of receptor
      - active cells: macrophages, histiocytes (reticuloendothelial system); Kupffer cells (macrophages)
      - atherosclerosis: foam cells in plaques derived from macrophages in the scavenger cell pathway
      - most cholesterol is still removed using the classic LDL receptor-mediated endocytosis
- HDL function 1: surface-lipid acceptor in chylomicron, VLDL catabolism
   - HDL: formed from nascent HDL precursors
      - synthesis: may be directly synthesized by the liver
      - catabolism: may form from surface coat of chylomicrons during chylomicron catabolism
   - triglyceride removal from chylomicron, VLDL cores requires surface shrinking; done through HDL maturation
   - lecithin:cholesterol acyl transferase (LCAT)
      - function: esterifies cholesterol to prevent transfer back to VLDL, chylomicrons
      - mechanism: transfers fatty acyl group from phospholipid (lecithin) to 3-OH of cholesterol, both at the surface
- HDL function 2: exchange of essential apoproteins between lipoprotein functions
   - CM’, VLDL’: immature lipoproteins
   - require transfer of specific apoproteins to, from HDL to complete metabolism and maturation
- HDL function 3: reverse cholesterol transport
   - free cholesterol in membrane bilayers is exchangable
   - LCAT: helps keep cholesterol locked into the lipoprotein
   - nascent HDL, small spherical HDL: can remove excess cholesterol from tissue plasma membranes
   - spherical HDL: transports the excess cholesterol ester to the liver for catabolism and excretion
      - direct process: receptor-mediated endocytosis, catabolism of secondary lysosome
      - indirect process: HDL transfer to VLDL, via cholesterol ester transfer protein
                                                                         Biochemistry: NOTES & OBJECTIVES (page 92 of 165)



       - cholesterol initially derived from excess extrahepatic free cholesterol
       - metabolized to LDL before transferal to liver


Notes: Lecture and Reading
structure of cholesterol
- carbon backbone: 27C
- forms
   - free form: unesterified, with alcohol group on C3; most common form in membranes
   - esterified form: fatty acid chain ester-linked to C3; storage form, present as intracellular lipid droplets
- acyl-CoA:cholesterol acyl transferase (ACAT): catalyzes esterification of free cholesterol
sources of cholesterol
- dietary intake
   - source: animal tissue, especially eggs, dairy products, and meat; less common in poultry, fish
   - prevalence: varies with diet
   - plants: lack cholesterol, but make ergosterol, an analog sterol
- synthesis
   - source: de novo synthesis from acetyl-CoA
   - prevalence: 60%, even in a high cholesterol diet (average: 300 mg via intake, 800 mg via synthesis)
synthesis of cholesterol
- distribution
   - normal dietary intake: primary source is liver, though synthesis probably occurs in all tissues except RBCs
   - low dietary intake: small intestine becomes a major producer in order to help with chylomicron synthesis
   - much of the liver cholesterol is exported to the blood in lipoproteins, or converted to bile acids
- carbon source: acetyl-CoA
   - all carbon in cholesterol is derived from acetyl-CoA
   - initial steps: same as ketone body synthesis
      - formation of 3-hydroxy-3-methyl-glutaryl-CoA, or HMGCoA)
      - note that ketone body synthesis occurs only in the liver, while cholesterol synthesis occurs in all tissues
   - localization: HMGCoA
      - ketone body formation: HMGCoA pool in mitochondria
      - cholesterol synthesis: HMGCoA pool in cytoplasm
- HMGCoA reductase: committed step in cholesterol synthesis
   - reaction:        HMGCoA  mevalonate [2 (NADPH  NADP+);  CoA-SH]
   - mechanism: reduction of thioester group to an alchohol, using 2 NADPH
   - energetics: controlled, committed step of cholesterol synthesis
- cholesterol formation from mevalonic acid: three additional phases (SUPPLEMENTARY)
   - formation of 5-carbon precursors: dimethylallyl pyrophosphate; isopentenyl pyrophosphate
   - head to tail condensation of the 5-carbon precursors, driven by pyrophosphate cleavage
      - C5 + C5  C10 + C5  C15
      - C15 + C15  C30
      - note that the C10 and C15 isoprenoid polymers are also used as membrane protein anchors
   - cyclization of the C30 squalene to form the four rings of cholesterol, generating 3 CO 2
conversion of cholesterol to bile acids
- synthesis and secretion of bile acids
   - primary bile acids: cholic acid, chenodeoxycholic acid (synthesized in the liver from cholesterol)
   - conjugation: both primary and secondary bile acids
      - carboxyl groups conjugated in amide linkage with either glycine or taurine before release into bile duct
      - increases hydrophobic character
   - modification
      - in the intestine, conjugated acids are hydrolyzed to regenerate the free carboxyl group, glycine, and taurine
      - secondary bile acids: some primary bile acids modified by bacteria, resulting in loss of 7-hydroxy group
- enterohepatic circulation: primary means of getting rid of cholesterol
   - major components and molar ratio
      - bile acids:                              16
      - phosphatidylcholine (“lecithin”):        3
                                                                         Biochemistry: NOTES & OBJECTIVES (page 93 of 165)



      - cholesterol:                               1
   - components are synthesized in the liver, stored in the gall bladder
   - cholesterol and bile acids
      - cholesterol is highly insoluble
      - requires bile acids to emulsify the cholesterol and form mixed micelles with lecithin
      - cholestasis: cholesterol proportion too high, resulting in insoluble gall stones and obstruction of the bile duct
   - circulation
      - circulated through intestine, absorbed by ileum, returned to liver via portal vein
      - 15-30 g of circulation (2-4 g pool of bile acids, which circulates 5-10 times daily)
   - reabsorption: both primary and secondary bile acids
   - excretion
      - bile acids: 0.2-0.6 g/day pass into feces, representing the major means of cholesterol excretion
      - cholesterol: 1 g secreted into intestine; only about 0.5 g reabsorbed
      - net steroid secretion: bile acids + cholesterol = approximately 1 g
- other components of the bile
   - bilirubin: breakdown product of heme catabolism; transported to liver via albumin forexcretion
   - biliary obstruction: can lead to accumulation of bilirubin in the liver, ultimately in the blood
   - jaundice: accumulation of bilirubin or bilirubin diglucouronide in the blood, causing yellow discoloration of skin
cholesterol absorption from the diet
- balance: Western societies
   - ingestion:       500 – 800 mg cholesterol per day
   - absorption: 250 – 400 mg cholesterol per day
   - synthesis:       1000 mg (1 g) cholesterol per day
                      - decline in diet: increase in de novo synthesis
                      - increase in diet: decline in de novo synthesis
- process of absorption: similarities to triglyceride
   - emulsification: dietary cholesterol esters, free cholesterol emulsified by bile acids, forming mixed micelles
   - hydrolysis: intestinal cholesterol esterase of emulsified cholesterols, forming free cholesterol
   - absorption: free cholesterol is absorbed into the mucosal cells of the small intestine
   - chylomicrons: some free cholesterol esterified, incorporated along with free cholesterol, triglycerides
   - transport: chylomicrons passed into lymph, dump off triglycerides in targets, and form chylomicron remnants
- chylomicron remnant (CMR): endocytosis into liver
   - CMR taken in by CMR receptor-mediated endocytotic process, catabolized
   - cholesterol esters on the remnant are hydrolyzed
   - free cholesterol: can be reesterified and stored, incorporated into lipoproteins, or converted to bile acids
- fat soluble vitamins follow a similar route of absorption
   - fat soluble vitamins: A, D, E, K
      - must be emulsified with other dietary fat to be absorbed by the intestinal mucosa
      - once absorbed, are packed on chylomicrons for transport to liver via CMR
   - interference in bile acid secretion can ultimately lead to fat soluble vitamin deficiency
- effects of diet
   - HMGCoA reductase: primary point of control
   - transcriptional control
      - regulation: sterol regulatory element (SRE) and binding protein (SREBP)
      - high cholesterol: binds, inactivates SREBP
      - high insulin: activates SREBP, increasing HMGCoA reductase and other lipogenic enzymes
   - dietary control
      - low dietary cholesterol
         - effect: increased HMGCoA reductase activity
         - because of this, severely limiting cholesterol intake leads to only modest reduction of plasma cholesterol
      - high dietary cholesterol
         - effect: decreased HMGCoA reductase activity
         - cholesterol taken up by liver as chylomicron remnants (or LDL) will cause the decrease in activity
      - caloric intake
         - insulin: promotes enzyme activation, increased synthesis
         - because of this, hepatic VLDL synthesis is stimulated by excess caloric intake
                                                                          Biochemistry: NOTES & OBJECTIVES (page 94 of 165)



         - excess alcohol or chronically high circulating FA also stimulate VLDL synthesis
      - fatty acid ration
         - increased polyunsaturated/saturated: decrease in cholesterol
         - mechanism: inhibit activation of SRE-mediated transcriptional events
         - note that this is independent of the correlation between saturated fatty acids and intrinsic cholesterol in food
   - summary: key points
      - high carbohydrate: increased plasma cholesterol
      - high saturated fat: increased plasma cholesterol
central role of liver
- liver: plays a key role in cholesterol homeostasis throughout the body, including plasma homeostasis
   - processing of dietary cholesterol            (absorbed as chylomicron remnants)
   - excretion of cholesterol                     (unabsorbed bile acids, cholesterol secreted in the bile)
   - maintenance of plasma cholesterol            (lipoproteins secreted by the liver, VLDL  LDL)
   - processing of excess cholesterol             (endocytosis of mature HDL, can be excreted or recirculated)
lipoprotein metabolism: summary
- exogenous pathway of cholesterol absorption
   - chylomicron maturation
   - lipoprotein lipase
   - CMR endocytosis
- endogenous pathway
   - VLDL’ export from liver
   - maturation into VLDL
   - catabolism to LDL via lipoprotein lipase
   - LDL endocytosis, by target or hepatic tissues
- reverse transport
   - use of nascent HDL
   - LCAT esterification, transfer of excess cholesterol, generating small, larger HDL
   - endocytosis of HDL
- key points
   - dietary fat: drives chylomicron synthesis and cholesterol “input” (exogenous pathway)
   - dietary CHO: drives VLDL synthesis (endogenous pathway)
   - density
      - triglycerides are relatively lighter than proteins
      - removal of triglycerides causes a relative increase in density
transfer of cholesterol to extrahepatic (non-liver) tissues
- overview
   - lipoproteins: form by which absorbed or synthesized cholesterol is returned to the liver
   - VLDL: used to transport triglycerides from liver to adipose, giving off LDL
   - LDL is the major source of cholesterol for non-hepatic tissues
      - post absorptive state (12 h fast): 2/3 of total plasma cholesterol carried on LDL
      - LDL carried to and metabolized by numerous tissue, including liver
      - de novo synthesis inhibited as LDL cholesterol from LDL is taken up
- classic LDL receptor-mediated endocytosis
   - binding
      - LDL binds high affinity receptor localized in clatharin coat pits on plasma membrane
      - internalized by endocytosis
   - endosome maturation
      - receptor returned to cell surface
      - endosome fuses with lysosome
      - lipoproteins degraded to monomeric constituents (cholesterol, fatty acids, amino acids, etc.)
   - cytoplasmic free cholesterol: can be placed in cell membranes or used in other synthetic reactions
   - excess free cholesterol
      - esterified by acylCoA:cholesterol acyl transferase (ACAT) [FACoA + cholesterol  chol. E + HSCoA]
      - stored in lipid droplets
   - regulation
      - HMGCoA reductase: feedback inhibition by cholesterol internalized by this mechanism
                                                                         Biochemistry: NOTES & OBJECTIVES (page 95 of 165)



      - LDL receptors: reduced in number by cholesterol feedback
      - ACAT: activated by excess cholesterol
- scavenger cell pathway
   - plasma LDL
      - has a half life of 12-24 hours
      - up to 2/3 of LDL removed from plasma done via the classical LDL receptor pathway
      - 1/3 or more of LDL removed via scavenger cell pathway
   - scavenger cell pathway
      - not well characterized; known to utilize another type of receptor
      - active cells: macrophages, histiocytes (reticuloendothelial system); Kupffer cells (macrophages)
      - atherosclerosis: foam cells in plaques derived from macrophages in the scavenger cell pathway
lipoprotein metabolism and the role of HDL
- overview: roles of HDL
   - regulation: promotes catabolism of chylomicrons and VLDL
   - reverse transport: carries excess cholesterol from extrahepatic tissues to the liver for excretion
- origin of HDL
   - mature HDL: formed from nascent HDL precursors
      - synthesis: may be directly synthesized by the liver
      - catabolism: may form from surface coat of chylomicrons during chylomicron catabolism
   - nascent HDL
      - morphology: disc-shaped
      - composition: phospholipids, cholesterol in a bilayer, complexed with apoproteins
      - normally not observed in plasma in people with normal LCAT function
   - lecithin:cholesterol acyl transferase (LCAT)
      - function: conversion of nascent HDL to spherical HDL
         - surface molecules (cholesterol, phospholipids, larger apoproteins) transferred to nascent HDL
         - LCAT produces cholesterol esters that will not transfer back
         - with accumulation, nascent HDL is converted to spherical HDL, with a cholesterol ester core
      - mechanism
         - transfers fatty acyl group from phospholipid (lecithin) to 3-OH of cholesterol, both at the surface
         - cholesterol migrates to the interior of the lipoprotein particle
- role of HDL in lipid transfer during chylomicron and VLDL catabolism
   - structure: chylomicrons, VLDL
      - core: numerous triglycerides, smaller amount of cholesterol ester
      - surface: apoproteins, phospholipid, cholesterol
   - lipid transfer
      - with triglyceride removal, core shrinks, and surface space must be reduced as well
      - surface molecules transferred to HDL particles, with resultant increase in HDL size
      - with increase in HDL surface particles, hydrophobic core must increase in size as well
      - core molecules are esterified by lecitin:cholesterol acyl-transferase (LCAT)
   - process: similar to that of conversion of nascent HDL to spherical HDL
- role of HDL in protein transfer during chylomicron and VLDL maturation
   - CM’
      - chylomicron secreted by intestinal mucosa
      - composition: triglyceride core, shell of phospholipid, cholesterol, and apoproteins
      - apoproteins: AI, AII, AIV, B48
   - CM
      - generated from apoprotein exchange with HDL
         - CM’: receives apoCII, apoE
         - spherical HDL: receives apoAI, apoAII
      - apoprotein functions
         - apoCII: activator of lipoprotein lipase
         - apoE: protein recognized along with apoB by the hepatic CMR receptor
   - VLDL’
      - lacks apoCII
      - must receive apoCII from spherical HDL before VLDL can be catabolized by lipoprotein lipase
                                                                       Biochemistry: NOTES & OBJECTIVES (page 96 of 165)



- role of HDL and LCAT in reverse cholesterol transport
   - free cholesterol in membrane bilayers is exchangeable
      - LCAT: helps keep cholesterol locked into the lipoprotein
      - deficiencies: net transfer of cholesterol from free-cholesterol enriched plasma lipoproteins to membranes
   - nascent HDL, small spherical HDL: can remove excess cholesterol from tissue plasma membranes
      - cholesterolmembrane ↔ cholesterolHDL  cholesterol esterHDL [LCAT]
      - LCAT forces equilibrium to right, resulting in cholesterol removal
   - spherical HDL: transports the excess cholesterol ester to the liver for catabolism and excretion
      - direct process: receptor-mediated endocytosis, catabolism of secondary lysosome
      - indirect process: transfer to VLDL
         - transfer occurs via a plasma cholesterol ester transfer protein
         - after conversion of VLDL to LDL, would be transferred to liver
deficiencies in lipoprotein metabolism
- dyslipidemia: major elevation or reduction of one or more of the major classes of lipid proteins
   - hyperlipoproteinemias: most common
      - originally classified based on whether triglyceride, cholesterol, or both are elevated
      - now further typing based on the class of lipoproteins (e.g.chylomicrons, VLDL)
   - hypercholesterolemias
      - cause: usually by elevated LDL, VLDL, with the LDL having the highest cholesterol content
      - elevated VLDL: would also cause elevated total plasma triglyceride
cholesterol and atherosclerosis
- atherosclerosis: accumulation in arterial tunica intima of plaque-like lipids which undergo calcification
- patterns
   - strong correlation between circulating cholesterol / frequency of atherosclerosis, coronary heart disease (CHD)
   - stronger correlation with LDL; inverse correlation with HDL
- clinical considerations
   - high HDL seems to prevent atherosclerosis
   - increase in HDL with decrease in LDL slows progression of atherosclerosis, sometimes even reversing it
- Tangier disease
   - defect in ABC1 transporter (transport out of cholesterol)
   - symptoms: yellow throat, caused by fatty/cholesterol accumulation
   - discovered in 1961; cause discovered in 1999

TABLE: Function of Select Lipoproteins
 apoprotein disease where role or function                                      defective
 AI         - major component of HDL
            - activator of LCAT
 B48 and    - major structural component of chylomicrons (B48),                 - abeta or hypobeta
 B100       VLDL (B100), LDL (B100)                                             - lipoproteinemia
            - protein recognized by LDL receptor
 CII        - present on mature chylomicrons and VLDL                           - familial apoCII deficiency
            - activator lipoprotein lipase
 CIII       - major component of chylomicrons, VLDL, and IDL
            - prevents recognition of B and E rich lipoproteins by
            hepatic CMR receptor and LDL receptor
 E          - required with aboB on chylomicron remnant for                     - familial type 3 hyperlipoproteinemia
            recognition by hepatic “remnant” receptor

TABLE: Lipoprotein Compositions
 type                    protein (%)        cholesterol (%)                phospholipid     triglyceride   mol. wt.
                                            free              ester        (%)              (%)
 chylomicrons (ULDL)           2            3                 4            6                85             108 - 109
 very low density (VLDL)       10           5                 8            12               65             107 - 108
 low density (LDL)             20           10                42           20               8              2-4 x 106
 high density (HDL)            46           4                 20           25               5              2-3 x 105
                                                                        Biochemistry: NOTES & OBJECTIVES (page 97 of 165)




44., 45. Regulation of Carbohydrate and Fat Metabolism in the Fed
         and Fasted States
Study Guide
know the following:
- direction of metabolism
   - FED:          glycolysis, pyruvate oxidation, glycogenesis, lipogenesis
   - FASTED:       gluconeogenesis, glycogenolysis, lipolysis, fatty acid oxidation
- know the key factors governing metabolic direction


Notes: Lecture and Reading
REGULATION OF METABOLISM IN THE CARBOHYDRATE FED STATE
- overview
   - status
      - glucose available for all cells to use
      - excess glucose should be stored as glycogen, fat
      - insulin/glucagon: high
         - cAMP low
         - protein kinase A enzymes will be dephosphorylated
   - active pathways in liver (or adipose)
      - glycolysis, pyruvate dehydrogenase: provides acetyl-CoA to TCA, maintains high ATP, acts in lipogenesis
      - glycogenesis:                            stores glucose as glycogen (primarily in liver and muscle)
      - pentose phosphate pathway:               NADPH for fatty acid synthesis
      - citrate shuttle:                         cytoplasmic acetyl-CoA, NADPH for fatty acid synthesis
      - fatty acid synthesis:                    acetyl-CoA carboxylase, fatty acid synthase
regulation
- factors governing glucose oxidation
   - glucose entry, phosphorylation: initial steps of glycolysis
      - glucokinase (liver): synthesis induced by insulin
      - GLUT-4: number in membrane increased by insulin
   - phosphofructokinase: key control of glycolysis
      - active due to high fructose 2,6-bisphosphate
         - high I/G: low cAMP
         - low cAMP: active PFK-2, inactive FBPase-2 (dephosphorylated)
      - key control, promoting glycolysis despite high ATP (which would normally inhibit)
   - pyruvate kinase: final step of glycolysis
      - high I/G: low cAMP
      - low cAMP: active pyruvate kinase (dephosphorylated)
      - synthesis also induced by insulin
   - pyruvate dehydrogenase: initial step in TCA cycle, forming acetyl-CoA
      - active due to low acetyl-CoA (a feedback inhibitor)
      - low acetyl-CoA due to low fatty acyl-CoA
   - acetyl-CoA: funneled to TCA, rather than gluconeogenesis
      - citrate synthase: active due to low mitochondrial fatty acyl-CoA
         - increased fatty acid synthesis: high malonyl-CoA
         - high malonyl-CoA: inhibits CoA:carnitine acyl transferase (CCAT)
         - inhibited CCAT: low mitochondrial fatty-acyl-CoA
         - low fatty acyl-CoA: active citrate synthase
      - mitochondrial NADH
                                                                        Biochemistry: NOTES & OBJECTIVES (page 98 of 165)



         - with increased energy, mitochondrial NADH levels will rise
         - this will cause inhibition of isocitrate dehydrogenase
         - this results in an increase in citrate levels
- glucose 6-phosphate utilization
   - glycogen: synthesized as an energy store
      - glycogen synthase active, glycogen phosphorylase inactive (dephosphorylated)
      - dephosphorylated form: due to low cAMP
   - pentose phosphate pathway: used to provide NADPH to fatty acid biosynthesis
      - NADPH: used in fatty acid biosynthesis, causing decline
      - glucose 6-P dehydrogenase
         - inhibited by NADPH; low NADPH thus causes increase in activity
         - synthesis: induced by insulin
- fatty acid synthesis
   - citrate shuttle: used to give a steady supply of acetyl-CoA to fatty acid biosynthesis
      - citrate: accumulates in mitochondria due to slowed TCA cycle, spills into cytoplasm
      - ATP citrate lyase: synthesis promoted by insulin
      - NADP+-dependent malate DH: synthesis promoted by insulin
      - pyruvate carboxylase: activated by high acetyl-CoA (usually always high enough to activate)
   - acetyl-CoA carboxylase: key control point of fatty acid synthesis, resulting in increased malonyl-CoA
      - activated by insulin-dependent phosphorylation
      - activated by reversal of cAMP-dependent phosphorylation (constitutive phosphoprotein phosphatase)
      - induced by high insulin
   - fatty acid synthase: induced by insulin
- triglyceride synthesis and storage
   - CCAT: functions to import fatty acids to mitochondria
      - inhibited by high malonyl-CoA
      - prevents oxidation, diverting fatty acids by mass action into triglyceride synthesis
   - glycerol 3-P: used for triglyceride backbone
      - glucose provided from outside tissue via GLUT-4
      - high GLUT-4: increased number in membrane due to high insulin
   - lipoprotein lipase: hydrolyzes triglycerides from VLDL, CM
      - activated by insulin
      - results in free fatty acids, fatty acid uptake and storage in adipose tissue
   - hormone-sensitive lipase: mobilizes triglycerides in adipose tissues
      - high insulin: leads to low cAMP
      - low cAMP: prevents lipolysis via hormone-sensitive lipase

REGULATION OF METABOLISM IN THE CARBOHYDRATE FASTED STATE
- overview
   - status
      - blood glucose initially maintained by glycogenolysis, then by gluconeogenesis
      - insulin/glucagon: low
         - cAMP high
         - protein kinase A enzymes will be phosphorylated
   - active pathways in liver (or adipose)
      - lipolysis:                     provides fatty acid (major metabolic fuel) and glycerol for gluconeogenesis
      - fatty acid oxidation:          maintains high cellular ATP, ketone body synthesis
      - glycogenolysis:                early in fast, maintains glucose levels in blood
      - gluconeogenesis:               later in fast, maintains glucose levels in blood
      - protein catabolism:            provides amino acid substrates for gluconeogenesis
regulation
- factors governing fat catabolism
   - hormone-sensitive lipase: key control of lipolysis (fat mobilization), increasing FA and glycerol in blood
      - low I/G: high cAMP
      - high cAMP: active protein kinase A
      - active protein kinase A: active hormone-sensitive lipase (phosphorylated)
                                                                      Biochemistry: NOTES & OBJECTIVES (page 99 of 165)



   - acetyl-CoA carboxylase: key control point of fatty acid synthesis, functioning in malonyl-CoA synthesis
      - liver, adipose: take in fatty acids from blood
      - low I/G: high cAMP
      - high cAMP: inactive acetyl-CoA carboxylase (cAMP-dependent phosphorylation)
   - NADH: maintained throughout fast
      - ATP/ADP: high due to fatty acid oxidation
      - NADH/NAD+: high due to fatty acid oxidation
      - the rate of fatty acid oxidation is limited only by the rate of electron transport
      - the rate of electron transport is governed by availability of NAD +, FAD, ADP
   - citrate synthase: key TCA enzyme, inhibited during fast
      - reduced fatty acid synthesis (inhibited acetyl-CoA carboxylase): low malonyl-CoA
      - low malonyl-CoA: active CCAT
      - active CCAT: increased fatty acyl-CoA
      - increased fatty acyl-CoA: inhibited citrate synthase
      - inhibited citrate synthase: increased acetyl-CoA in mitochondria
   - ketone body synthesis: results from increased acetyl-CoA
- factors governing glucose output by liver
   - time course
      - early: use of glycogen to maintain glucose
      - late: use of gluconeogenesis to maintain glucose
   - glycogen
      - glycogen phosphorylase active, glycogen synthase inactive (phosphorylated)
      - phosphorylation: low I/G gives high cAMP gives high protein kinase A
   - substrates for gluconeogenesis
      - glycerol: lipolysis
      - amino acids: released from muscle protein degradation, with muscle uptake inhibited by low I/G
   - pyruvate: diverted from TCA cycle to oxaloacetate and gluconeogenesis
      - pyruvate dehydrogenase: inhibited by high acetyl-CoA
      - PEP carboxylase: activated by cAMP (low I/G)
      - pyruvate kinase: inactivated by cAMP (protein kinase A-mediated phosphorylation)
   - fructose 1,6-bisP  fructose 6-P
      - inactive due to low fructose 2,6-bisphosphate
         - low I/G: high cAMP (PKA)
         - high cAMP: active FBPase-2, inactive PFK-2 (phosphorylated)
      - key control, inhibiting glycolysis along with high ATP
   - glucose 6-P  glucose
      - glucose 6-P phosphatase: activated (mechanism unknown)
      - glucokinase: synthesis slowed (lack of induction by insulin)
- glucose sparing by muscle
   - low I/G: inhibits transport in
   - fatty acids: maintain ATP, preventing glucose oxidation by controls discussed in Module 3

KEY FACTORS GOVERNING DIRECTION OF METABOLISM DURING FEEDING AND FASTING
feeding: insulin (I/G is high)
- enzymes induced by insulin
   - glycolysis:            glucokinase, pyruvate kinase
   - pentose phosphate:     glucose 6-P dehydrogenase
   - fatty acid synthesis: acetyl-CoA carboxylase, fatty acid synthase
   - citrate shuttle:       ATP-citrate lyase, NADP+-dependent malate dehydrogenase
- enzymes activated by insulin
   - lipoprotein lipase              (mechanism unknown)
   - acetyl-CoA carboxylase          (insulin-dependent kinase)
- enzymes activated or inactivated due to lack of cAMP-dependent phosphorylation
   - glycogen synthase:              A
   - glycogen phosphorylase:         I
   - phosphofructokinase-2:          A
                                                                     Biochemistry: NOTES & OBJECTIVES (page 100 of 165)



   - fructose bisphosphatase-2:       I
   - pyruvate kinase:                 A
   - acetyl-CoA carboxylase:          A
   - hormone-sensitive lipase:        I
   - HMGCoA reductase:                A
- allosteric effectors changing in level
   - cAMP:                   decreased      inactive PKA; due to low I/G
   - fructose 2,6-bisP:      increased      activates PFK-1, inactivates FBPase-1
   - malonyl-CoA:            increased      inhibits CCAT
   - acetyl-CoA:             decreased      leads to increased pyruvate DH activity
fasting: glucagon (I/G low)
- enzymes induced
   - PEP carboxykinase: gluconeogenesis
   - glucose 6-phosphatase: gluconeogenesis
- enzymes activated or inactivated by cAMP-dependent phosphorylation
   - glycogen synthase:               I
   - glycogen phosphorylase:          A
   - phosphofructokinase-2:           I
   - fructose bisphosphatase-2:       A
   - pyruvate kinase:                 I
   - acetyl-CoA carboxylase:          I
   - hormone-sensitive lipase:        A
   - HMG-CoA reductase:               I
- allosteric effectors changing in level
   - cAMP:                   increased      activates PKA; due to low I/G
   - fructose 2,6-bisP:      decreased      inactivates PFK-1, activates FBPase-1
   - malonyl-CoA:            decreased      leads to increased CCAT activity
   - acetyl-CoA:             increased      inhibits pyruvate DH
   - fatty acyl-CoA          increased      inhibits citrate synthase




46. Protein and Amino Acid Metabolism – Overview and Central
        Reactions
Study Guide
know the following:
- protein turnover
   - 70 kg adult: ~300 g body protein degraded, resynthesized daily (know this number)
   - protein half lives: vary greatly, on average from 1 hour to 1 week
   - 15-20% of energy in basal metabolic rate used to provide NTPs for the resynthesis process
      - during fasting, degradation does not change greatly – still used in gluconeogenesis
      - normal resynthesis is reduced, so energy must be conserved
- essential amino acids
   - all 20 amino acids are simultaneously required for normal protein synthesis
   - amino acids are not stored, and even essential amino acids may be broken down in the absence of synthesis
   - one must therefore continually take in amino acids, especially essential amino acids, in the diet
- preferential catabolic use
   - liver:     Ala
   - muscle: Val, Ile, Leu              (exports Ala, Gln)
   - kidney: Gln                        (esp. in late fasting for gluconeogenesis)
- amino acid catabolism: central reactions
   - transamination: AA aminotransferase
      - reaction: AA + α-ketoglutarate  α-keto acid + glutamate
                                                                       Biochemistry: NOTES & OBJECTIVES (page 101 of 165)



     - enzyme: AA aminotransferase
     - cofactor: [PLP]
     - energetics: reversible; can be used to generate an amino acid from a given α-keto acid
     - use:        initial step in catabolism of numerous amino acids, such as Ala or Asp
  - oxidative deamination: glutamate dehydrogenase
     - reaction: glutamate  α-ketoglutarate [NAD+  NADH + H+; H2O  NH3]
     - enzyme: glutamate dehydrogenase
     - energetics: reversible; can be used to generate glutamate from α-ketoglutarate
     - use:        frequently coupled to deamination, generating a substrate for urea synthesis
  - glutamine formation: glutamate synthetase
     - reaction: glutamate  glutamine [NH3 ; ATP  ADP + Pi]
     - enzyme: glutamine synthetase
     - use:        glutamine is a donor in biosynthesis, and is also used as a transport vehicle for NH 3
  - glutamate formation: glutaminase
     - reaction: glutamine  glutamate [H2O  NH3]
     - enzyme: glutaminase
     - use:        often used in kidney to break down glutamine used in ammonia transport


Notes: Lecture and Reading
overview of protein and amino acid metabolism
- amino acid pool
   - proteins in constant degradation/synthesis cycle, and all 20 amino acids needed for synthesis of most proteins
   - essential amino acids: those eight amino acids which cannot be synthesized and must be obtained in the diet
   - amino acid pool: total mixture of amino acids within all cells, with individual cellular pools connected by blood
- homeostasis
   - protein degradation and diet: add to pool
   - synthesis, metabolism:             subtract from pool
   - to maintain homeostasis, it is imperative to replace essential amino acids in the pool
protein turnover and need for dietary amino acids
- essential amino acids
   - essential amino acids must be provided for in diet
   - tend to be the more structurally complex ones
      - aliphatic: Val, Ile, Leu
      - sulfur:     Met
      - alcohol:    Thr
      - basic:      Lys, *His, *Arg
      - aromatic: Trp, Phe
   - His and Arg
      - required only in diet of infant or growing child
      - can be synthesized, but not in quantities sufficient to support rapid growth
- minimum dietary protein requirement
   - minimum daily requirement: 0.8 g per kg body mass (56 g for 70 kg adult)
   - dietary recommendation:            12-15% of total caloric intake (90-110 g)
   - infants < 3 months:                2.4 g/kg
                6 months:               1.5 g/kg
                1 year:                 1.3 g/kg
   - varies with nutritional quality of protein
      - vegetable proteins: typically deficient in Lys, Met
      - can be overcome through careful consumption (cereal has Met, legumes have Lys)
      - however, one needs amino acids at the same time, so genetic engineering can aid this
protein turnover
- use
   - 70 kg adult:                       ~300 g body protein degraded, resynthesized daily (know this number)
      - muscle myofibrillar protein: 100 g/day
      - digestive enzymes:              50 g/day
                                                                        Biochemistry: NOTES & OBJECTIVES (page 102 of 165)



      - intestinal epithelium:         20 g/day
   - 15-20% of energy in basal metabolic rate used to provide NTPs for the resynthesis process
- protein half lives: vary wildly (typically ~1 hour to ~ 1 week)
   - inducible:               usually have shorter half lives
   - other proteins:          considerably more stable
                              - blood plasma proteins: 10 days
                              - RBC proteins:             120 days
                              - muscle myosin:            180 days
- fasting state
   - gluconeogenesis: requires ~75 g/day
      - degradation:          does not change
      - resynthesis:          decreased
   - because of 15% use for energy, one must conserve energy during fasting state
digestion of dietary protein and absorption of amino acids
- proteolysis
   - stomach
      - HCl:                  secretions from parietal cells that lower pH to approximately 1
      - pepsinogen:           secretory cell secretion that is activated to pepsin by HCl; begins proteolysis
   - intestine
      - bicarbonate:          pancreatic secretion that neutralized pH
      - proteases:            pancreatic secretions that hydrolyze proteins to amino acids, oligopeptides
      - mucosa cells:         absorb amino acids, oligopeptides, and complete proteolysis to amino acids
- utilization of amino acids by different tissues
   - tissues preferentially use certain amino acids for catabolism
   - intestinal mucosa:       Glu, Gln, Asp, Asn; exports Ala
   - liver:                   Ala
   - muscle:                  branched chain (Val, Ile, Leu); exports Ala (taken by liver), Gln (taken by kidney, liver)
   - kidney:                  Gln (deamidinated to Glu, releasing NH3); Glu converted to Ala and exported
central reactions and role of glutamate
- amino acid transaminase: transamination
   - reaction:       α-AA + α-ketoglutarate  α-keto acid + glutamate [PLP]
   - enzyme:         amino acid transaminase
   - mechanism: transamination (amino, keto groups essentially switched)
   - energetics: reversal; requires pyridoxal phosphate as a cofactor
   - use:            ALL transaminases use α-ketoglutarate and glutamate as a substrate/product pair
- glutamate dehydrogenase: oxidative deamination
   - reaction:       glutamate  α-ketoglutarate [H2O  NH3; NAD+  NADH + H+]
   - enzyme:         glutamate dehydrogenase
   - mechanism: deamination, replacement with hydroxyl group; reduction to ketone
   - energetics: reversible; often coupled to transaminations
   - location:       mitochondrial matrix
- glutamate synthetase and glutaminase
   - glutamine synthetase
      - reaction: glutamate  glutamine [NH3 ; ATP  ADP + Pi]
      - enzyme: glutamine synthetase
   - glutaminase
      - reaction: glutamine  glutamate [H2O  NH3]
      - enzyme: glutaminase
   - purpose: Gln can be used as a transport vehicle for NH3 in the blood, as in nitrogen excretion
- pyridoxine (vitamin B6)
   - solubility:              water-soluble
   - enzyme cofactor:         pyridoxal phosphate (PLP) or pyridoxamine phosphate
   - distribution:            transaminase, dehydratase reactions
- glutamine as N-donor in purine, pyrimidine synthesis (SUPPLEMENTARY)
   - Gln  PRPP  purines
   - Gln  carbamoyl-P [CP synthetase II]  pyrimidines
                                                                    Biochemistry: NOTES & OBJECTIVES (page 103 of 165)




47. Amino Acid Catabolism – Excretion of Nitrogen as Urea and C-
       Skeleton Metabolism
Study Guide
know the following:
- toxicity of ammonia
   - 90 % of nitrogen excreted from body is from amino acid catabolism
   - ammonia is toxic
      - α-ketoglutarate, a TCA cycle intermediate, is converted to Glu in the presence of excess ammonia
      - on certain tissues, especially the brain, this can be devastating
   - most excess ammonia is therefore converted to urea, a non-toxic product, prior to excretion
- ammonia generation
   - transamination: Ala, Asp, others
      - AA + α-ketoglutarate  α-KA + glutamate [AA aminotransferase, PLP]
      - glutamate  α-ketoglutarate [glutamate dehydrogenase: NAD+  NADH + H+; H2O  NH3]
   - nonoxidative dehydrogenation: Ser, Thr
      - serine  pyruvate [serine dehydratase, PLP:  NH3]
      - threonine  propionyl-CoA [threonine dehydratase, PLP:  NH3]
   - deamidination: Gln, Asn
      - glutamine  glutamate [glutaminase: H2O  NH3]
      - asparagine  aspartate [asparaginase: H2O  NH3]
- urea synthesis: ornithine and aspartate cycles
   - ornithine cycle: synthesis of 1 urea, using 4 ATP
      - * carbamoyl-P synthetase I:               NH3 + CO2  carbamoyl phosphate [2 ATP  2 ADP + 1 Pi ]
      - * ornithine transcarbamoylase:            ornithine + carbamoyl phosphate  citrulline [Pi]
      - arginosuccinate synthetase:               citrulline  arginosuccinate [aspartate ; ATP  AMP + PPi]
      - arginosuccinate lyase:                    arginosuccinate  arginine + fumarate
      - arginase:                                 arginine  ornithine [H2O  urea]
   - aspartate cycle: regeneration of aspartate
      - arginosuccinate synthetase:               aspartate  arginosuccinate [citrulline ; ATP  AMP + PPi]
      - arginosuccinate lyase:                    arginosuccinate  fumarate [ arginine]
      - fumarase:                                 fumarate  malate [H2O ]
      - malate dehydrogenase:                     malate  oxaloacetate [NAD+  NADH + H+]
      - aspartate aminotransferase:               oxaloacetate  aspartate [glutamate  α-ketoglutarate]
   - localization: * denoted reactions take place in the mitochondria
   - coupling: glutamate in AST reaction can be regenerated through an AA aminotransferase reaction
- urea synthesis: regulation
   - allosteric: N-acetylglutamate in response to a high protein diet
      - N-acetylglutamate: synthesized from glutamate, acetyl-CoA
      - increases in dietary protein lead to increases in N-acetylglutamate by an unknown mechanism
   - hormonal: glucagon in response to high protein diet
      - glucagon  increased cAMP  synthesis of the enzymes of the urea cycle
      - increases in dietary amino acids: promote glucagon release from α cells of the pancreas
- carbon skeleton metabolism: Phe
   - pathway
      - phenylalanine  tyrosine [phenylalanine hydroxylase]
      - tyrosine    fumarate, acetoacetate (Tyr also a precursor to catecholamines, which include adrenalin)
   - defect: phenylketonuria
      - problem: phenylalanine hydroxylase defects force metabolism to phenylpyruvate, phenyllactate
      - symptoms: poor neurological development
      - treatment: synthetic diet low in Phe, sufficient Tyr
                                                                   Biochemistry: NOTES & OBJECTIVES (page 104 of 165)



      - screening: routine due to prevalence of PKU births
- branched chain carbon skeleton metabolism: Val, Ile, Leu
   - common path
      - branched chain amino acid transaminase:            AA + α-ketoglutarate  α-KA + glutamate
      - branched chain α-keto acid dehydrogenase:          α-KA  R-CO-SCoA [HSCoA  CO2; NAD+ NADH, H+]
   - subsequent paths
      - Val, Ile    (glucogenic):                propionyl-CoA
      - Leu         (ketogenic):                 acetoacetate  acetyl-CoA
   - defect: maple syrup urine disease
      - problem: branched chain AA dehydrogenase defect causes accumulation of α-keto acids
      - symptoms: ketosis, neurological defects
      - treatment: synthetic diet restricted in branched chain amino acids
      - screening: no longer done due to rarity of disease
- propionyl-CoA metabolism: Thr, Met, Ile, Val
   - pathway
      - α-KB DH:                                α-ketobutyrate  propionyl-CoA
                                                  [TPP, lipoic acid, FAD: HSCoA  CO2; NAD+  NADH + H+]
      - propionyl-CoA carboxylase:              propionyl-CoA  D-methylmalonyl-CoA
                                                  [biotin: CO2 ; ATP  ADP + Pi]
      - racemase:                               D-methylmalonyl-CoA  L-methylmalonyl-CoA
      - methylmalonyl-CoA mutase:               L-methylmalonyl-CoA  succinyl-CoA
                                                  [B12]
   - defect: pernicious anemia and B12 deficiencies
      - problem: accumulation of methylmalonyl-CoA, which is neurotoxic (possibly competes with malonyl-CoA)
      - symptoms: often presents with anemia due to secondary folate deficiency (separate B 12 reaction)



Notes: Lecture and Reading
generation of NH3 during amino acid catabolism
- most amino acids are odidatively deaminated by coupling of their transamination with glutamate dehydrogenase
   - example: alanine deamination producing pyruvate
   - net reaction: alanine  pyruvate + NH3 + NADH + H+ [alanine aminotransferase, glutamate DH]
                    - ALT:             alanine + α-ketoglutarate  pyruvate + glutamate
                    - Glu DH:          glutamate  α-ketoglutarate [H2O  NH3; NAD+  NADH + H+]
- dehydratases release NH3 from Ser, Thr in a nonoxidative reaction
   - serine dehydratase:               serine  pyruvate [PLP: NH3]
   - threonine dehydratase:            threonine  α-ketobutyrate [PLP: NH3]
- NH3 is released during deamidination of Gln and Asn
   - glutaminase:                      glutamine  glutamate [H2O  NH3]
   - asparaginase:                     asparagine  aspartate [H2O  NH3]
excretion of nitrogen as urea
- nitrogen excretion
   - urea:          85% of excreted nitrogen
   - creatinine:    4-5 %
   - ammonia:       2-3 %
   - uric acid:     1-2 %
   - others:        5%
- ammonia toxicity
   - NH3: depletes TCA cycle of α-ketoglutarate through mass action effect on glutamate dehydrogenase
   - slowing of TCA cycle: leads to decreased rates of ATP synthesis, hepatic coma
   - nerve cells: particularly affected by excess NH3
- ornithine cycle: urea synthesis in the liver
   - carbamoyl-P synthetase I
      - reaction:             NH3 + CO2  carbamoyl phosphate [2 ATP  2 ADP + Pi ]
      - enzyme:               carbamoyl-P synthetase I
                                                                      Biochemistry: NOTES & OBJECTIVES (page 105 of 165)



      - regulation:            N-acetylglutamate: allosteric activation
      - location:              mitochondrial matrix
   - ornithine transcarbamoylase
      - reaction:              ornithine + carbamoyl phosphate  citrulline [Pi]
      - enzyme:                ornithine transcarbamoylase
      - location:              mitochondrial matrix (after ornithine transport in)
   - arginosuccinate synthetase
      - reaction:              citrulline  arginosuccinate [aspartate ; ATP  AMP + PPi]
      - enzyme:                arginosuccinate synthetase
      - energetics:            PPi  2 Pi
      - location:              cytoplasm (after citrulline transport out)
   - arginosuccinate lyase
      - reaction:              arginosuccinate  arginine + fumarate
      - enzyme:                arginosuccinate lyase
      - location:              cytoplasm
   - arginase
      - reaction:              arginine  ornithine + urea [H2O ]
      - enzyme:                arginase
      - location:              cytoplasm
   - net result: 1 urea synthesized, 4 ATP used in each turn of the urea cycle
- aspartate cycle: fumarate back to aspartate
   - fumarase:                           fumarate  malate [ H2O]
   - malate DH:                          malate  oxaloacetate [NAD+  NADH + H+]
   - aspartate aminotransferase:         oxaloacetate + glutamate  aspartate aminotransferase + α-ketoglutarate
   - AA aminotransferase:                α-AA + α-ketoglutarate  α-keto acid + glutamate
- regulation of urea synthesis
   - allosteric activation: N-acetylglutamate
      - N-acetylglutamate: synthesized from glutamate, acetyl-CoA
      - increases in dietary protein lead to increases in N-acetylglutamate (unknown mechanism)
   - hormonal activation: glucagon
      - glucagon  increased cAMP  synthesis of the enzymes of the urea cycle
      - dietary amino acids: promote glucagon release from α cells of the pancreas
- birth defects in urea cycle enzymes
   - severe urea cycle deficiency: 1 / 30,000 newborn infants
   - extent varies widely
      - those with complete deficiencies may die from hyperammonemia after first protein feeding
      - aggressive therapy must be begun immediately
entry of carbon chains into the central pathways of metabolism
- Phe: phenylalanine metabolism and phenylketonuria (PKU)
   - normal metabolism
      - phenylalanine hydroxylase: phenylalanine  tyrosine
      - transaminase:                    tyrosine  α-keto acid
                                         α-keto acid    fumarate, acetoacetate
   - importance
      - phenylalanine is an essential amino acid, tyrosine is not
      - phenylalanine is the means by which tyrosine is generated
   - defect: phenylketonuria
      - etiology: defect in phenylalanine hydroxylase
                     - dietary phenylalanine accumulates
                     - metabolized to phenylpyruvate, then to phenyllactate and phenylacetate, which accumulate
      - symptoms: neurological disorders, mental retardation
      - frequency: carriers: 1/50 to 1/60
                     afflicted: 1/10,000
      - screening: measurement of serum Phe levels after birth; required in Wisconsin screening programs
      - treatment: affected infants placed on a synthetic low-phenylalanine formula, diet
- Val, Ile, and Leu: branched chain amino acid metabolism
                                                                    Biochemistry: NOTES & OBJECTIVES (page 106 of 165)



   - common path metabolism: initial reactions carried out by enzymes recognizing all three amino acids
      - branched chain AA transaminase
         - reaction:           AA + α-ketoglutarate  α-keto acid + glutamate
         - enzyme:             branched chain amino acid transaminase
         - specificity:        Val, Ile, Leu
      - branched chain α-keto acid dehydrogenase
         - reaction:           α-keto acid  R-CO-SCoA [HS-CoA  CO2; NAD+  NADH + H+]
         - enzyme:             branched chain α-keto acid dehydrogenase
         - specificity:        Val, Ile, Leu
   - subsequent metabolism: glucogenic vs. ketogenic amino acids
      - glucogenic: can give rise to glucose
         - examples: Val, Ile
         - give rise to propionyl-CoA, which can give a net conversion to glucose
      - ketogenic: cannot be used to make glucose
         - examples: Leu
         - gives rise to acetyl-CoA, which cannot be used to make a net conversion to glucose
   - maple syrup urine disease
      - etiology: defect in branched chain α-keto acid dehyrdogenase, causing keto acid accumulation
      - symptoms: ketosis, neurological problems
      - frequency: afflicted: 1/250,000
      - screening: originally part of the Wisconsin screening program; no longer, due to low frequency
      - treatment: dietary restriction of branched chain amino acids
- propionyl-CoA metabolism and the role of vitamin B12
   - α-ketobutyrate dehydrogenase
      - reaction: α-ketobutyrate  propionyl-CoA [HSCoA  CO2; NAD+  NADH + H+]
      - enzyme: α-ketobutyrate DH
      - cofactors: TPP, lipoic acid, FAD
      - sources:      Thr, Met
   - propionyl-CoA carboxylase
      - reaction: propionyl-CoA  D-methylmalonyl-CoA [H2CO3 ; ATP  ADP + Pi]
      - enzyme: propionyl-CoA carboxylase
      - cofactors: biotin
      - sources:      Val, Ile
   - racemase
      - reaction: D-methylmalonyl-CoA  L-methylmalonyl-CoA
      - enzyme: racemase
   - methylmalonyl-CoA mutase
      - reaction: L-methylmalonyl-CoA  succinyl-CoA
      - enzyme: methylmalonyl-CoA mutase
      - cofactors: vitamin B12
      - mech.:        unique to human biochemistry, though others use vitamin B12




48. Methylation, One Carbon Transfer, Folate, and Vitamin B12
Study Guide
know the following:
- overview: methylation, folate, and B12
   - folate cycle
      - folate  dihydrofolate                       [folate reductase: NADPH + H+  NADP+]
      - dihydrofolate  tetrahydrofolate (THF)       [dihydrofolate reductase: NADPH + H+  NADP+]
      - tetrahydrofolate  5,10-methylene-THF        [PLP: Ser  Gly + H2O]
      - 5,10-methylene-THF  5-methyl-THF            [5,10-methylene-THF reductase: NADH + H+  NAD+]
                                                                    Biochemistry: NOTES & OBJECTIVES (page 107 of 165)



      - 5-methyl-THF  THF                               [B12: homocysteine  methionine]
   - S-adenosylmethionine cycle
      - homocysteine  methionine                        [B12: 5-methyl-THF  methionine]
      - methionine  S-adenosylmethionine                [ATP  PPi + Pi]
      - S-adenosylMet  S-adenosylhomocysteine           [ CH3 to methyl acceptors]
      - S-adenosylhomocysteine  homocysteine            [H2O  adenosine]
- S-adenosylmethionine: general methyl donor in cells, synthesized from ATP and Met
- folate
   - folate trap
      - 5,10-methylene-THF reductase: irreversible reaction generating 5-methyl THF
      - if the subsequent B12-dependent reaction is blocked, folate will become trapped as 5-methyl-THF
   - DNA synthesis
      - thymidilate synthase: dUMP  dTMP [5,10-methylene-THF  dihydrofolate]
      - dihydrofolate reductase: dihydrofolate  tetrahydrofolate
      - because of the use in DNA synthesis, dihydrofolate is a target of chemotherapeutic drugs
- homocysteine
   - can be metabolized to homocystine
   - can be metabolized to α-ketobutyrate via two PLP-dependent steps
- deficiencies
   - folate: homocystinuria resulting from accumulation of homocysteine due to lack of 5-methyl-THF
                 anemia resulting from poor thymidilate synthetase, immature blood cell production
   - B12:        homocystinuria resulting from accumulation of homocysteine due to lack of B 12
                 anemia resulting from secondary folate deficiency due to the folate trap
                 accumulation of methylmalonates resulting from lack of B 12-dependent mutase reaction


Notes: Lecture and Reading
overview
- important reagents
   - S-adenosylmethionine: general methyl donor in cells
   - vitamin B12: used in a reaction that regenerates S-adenosylmethionine
   - folate: derivative is a methyl donor to S-adenosylmethionine
- deficiencies
   - deficiencies in folate, B12 are common and of considerable medical significance
   - recent recommendation for significant folate supplements in American diet
- pathway
   - folate cycle
      - folate  H2-folate [folate reductase: NADPH + H+  NADP+]
      - H2-folate  H4-folate [dihydrofolate reductase: NADPH + H+  NADP+]
      - H4-folate  5,10-methylene-THF [PLP: serine  glycine + H2O]
      - 5,10-methylene-THF  5-methyl-THF [5,10-methylene-THF reductase: NADH + H+  NAD+]
      - 5,10-methyl-THF  H4-folate [ B12: homocysteine  Met ]
   - S-adenosylmethionine cycle
      - homocysteine  methionine [B12: 5-methyl-THF  THF]
      - methionine  S-adenosylmethionine [ATP + H2O  PPi + Pi]
      - S-adenosylmethionine  S-adenosylhomocysteine [CH3 to methyl acceptors]
      - S-adenosyl-homocysteine  homocysteine [H2O  adenosine]
S-adenosylmethionine and methylation
- S-adenosylmethionine
   - function:       general methyl donor in cells
   - synthesis:      condensation between S of methionine, 5’ ribose carbon of ATP
                     methyl group on S can be transferred to a series of acceptors
   - reactions:      S-adenosylmethionine  S-adenosylhomocysteine [CH3 to methyl acceptors]
                     hydrolysis:       S-adenosyl-homocysteine  homocysteine [H2O  adenosine]
                     methylation:      homocysteine  methionine [B12: 5-methyl-THF  THF]
                     adenosylation: methionine  S-adenosylmethionine [ATP + H2O  PPi + Pi]
                                                                    Biochemistry: NOTES & OBJECTIVES (page 108 of 165)



- methyl group acceptors from S-adenosylmethionine
   ACCEPTOR                               PRODUCT
   - guanidoacetate                       - creatine
   - phosphatidylethanolamine             - phosphatidylcholine
   - norepinephrine                       - epinephrine
   - protein-lysyl                        - methylated-lysyl, carnitine
   - rRNA and tRNA                        - methylated RNA
   - DNA                                  - methylated DNA
folate metabolism
- conversion of dietary folate to THF
   - folate:          derivatives serve as 1-C donors in numerous metabolic pathways
                      - de novo purine synthesis
                      - thymidylate synthesis
   - reactions:       folate reductase:             folate  H2-folate [NADPH + H+  NADP+]
                      dihydrofolate reductase: H2-folate  H4-folate [NADPH + H+  NADP+]
- serine donates one C to THF to generate 5,10-methylene-THF
   - reaction:        H4-folate  5,10-methylene-THF [PLP: serine  glycine + H2O]
   - cofactor:        PLP; often required in reactions involving amino acids
   - donor:           Ser often the donor, though other amino acids (Gly, His) can also serve as donors
- 5,10-methylene-THF is reduced to 5-methyl-THF
   - reaction:        5,10-methylene-THF  5-methyl-THF
   - enzyme:          5,10-methylene-THF reductase
   - energetics: irreversible: if vitamin B12 in short supply, all folate would become trapped as 5-methyl-THF
- thymidilate synthase generates DHF from 5,10-methylene-THF
   - reaction:        thymidilate synthase:         5,10-methylene-THF + dUMP  DHF + dTMP
                      dihydrofolate reductase: H2-folate  THF [NADPH + H+  NADP+]
   - clinical:        dihydrofolate reductase inhibitors may be used to inhibit cancer cell proliferation
                      - act by blocking regeneration of 5,10-methylene-THF, depriving cells of dTMP (and dTTP)
                      - examples: methotrexate, 5-fluorouracil
folate and B12 vitamin deficiency
- primary folate deficiency
   - megaloblastic anemia: prevalence of large, immature RBC precursors in the bone marrow and blood
      - cause: inhibition of normal DNA synthesis and cell division, due to lack of nucleotide precursors
      - folate required for de novo purine synthesis, conversion of dUMP to dTMP
   - homocystinuria: accumulation of homocysteine in the urine
      - cause: lack of 5-methyl-THF from folate deficieny
      - much of homocysteine nonenzymatically oxidized to homocystine
- primary B12 deficiency and secondary folate deficiency
   - B12 as an essential part of the diet
      - dependent on bacteria for vitamin B12 synthesis
         - plants do not use it, so strict vegetarians may eventually be at risk for deficiency
         - meat: general source, as other animals absorb it and use it in their metabolism
      - storage (liver): can store 6 year supply
      - only trace quantities are needed in diet
   - deficiency etiology
      - dietary extreme
      - inadequate absorption
         - “intrinsic factor”: glycoprotein of the gastric mucosa that is required for B 12 absorption
         - pernicious anemia: disease in which insufficient quantities of intrinsic factor are made
   - primary vitamin B12 deficiency
      - biochemical effects: accumulation of methylmalonate, homocysteine
      - methylmalonate: branched chain amino acid metabolism
         - causes severe neurological problems, demyelination
         - methylmalonyl-CoA may compete with malonyl-CoA in FA synthesis, causing abnormal branching
      - homocysteine: one carbon transfer reactions
         - appear to be less acutely damaging
                                                                       Biochemistry: NOTES & OBJECTIVES (page 109 of 165)



         - have been correlated with Alzheimer’s disease, vascular damage leading to atherosclerosis
   - secondary folate deficiency: folate trapped as 5-methyl-THF
      - megaloblastic anemia: found in B12 deficiencies, as in primary folate deficiencies
      - treatment: folate supplementation to overcome the anemia
         - does not correct the deficiency in the methylmalonyl mutase reaction
         - thus methylmalonate accumulation, the most dangerous aspect of B 12 deficiency, is not treated
vitamin B6 also plays an important role in homocysteine metabolism
- metabolism of homocysteine to α-ketobutyrate occurs in two B6 dependent steps
   - homocysteine + serine  cystathionine [PLP:  H2O]
   - cystathionine  α-ketobutyrate + cysteine [PLP: H2O  +NH4]
- secondarily generates cysteine from serine




49., 50. Renal Biochemistry and Function in Water, Electrolyte, and
         H+ Balance
Study Guide
part I – know the following:
- primary electrolyte composition of body fluids
   - intracellular anions:              K+, Mg2+
                    cations:            organic phosphates, proteins
   - extracellular: anions:             Na+
                    cations:            Cl-, HCO3-
- fluid balance
   - intracellular: cellular plasma membrane pumps (somewhat limited)
   - extracellular: kidney (extensive control of total volume, ion composition)
- kidney function
   - glomerular filtration: 125 mL/min (180 L/day), with composition similar to blood plasma minus proteins
   - ionic reabsorption: 99 % of Na+, Cl-, HCO3-, glucose, amino acids reabsorbed
   - water reabsorption: 99% of water reabsorbed, generating ~1L of hypertonic urine per day
   - creatinine: generally not reabsorbed, thus allowing one to estimate GFR through creatinine clearance
part II – know the following:
- acid-base balance: the mechanisms involved in maintaining H+ homeostasis and correction of imbalances
   - balance: maintained only when H+ output equals H+ produced (~70 meq/day)
   - input: can be altered significantly by diet and metabolism
      - salts:                alkalinizing effect        net proton consumption in catabolism
      - carboxylates:         alkalinizing effect        net proton consumption in catabolism
      - amino groups:         acidifying effect          net proton production in catabolism
      - inc. metabolism:      acidifying effect          H+ production in synthesizing lactate, ketone bodies
   - body buffers
      - types
         - hemoglobin, plasma protein
         - bicarbonate buffer
         - phosphate buffer (minor in plasma, but important in urine)
      - buffers can minimize the change in H +, but they do NOT correct the disturbance
- bicarbonate buffer
                                                             
                                    • HA  H  A
                                           a   K
  - Henderson-Hasselbach:

                                    • pH  pK a  log
                                                           A 
                                                              


                                                           HA 
          -
  - HCO3 equilibrium:                                                                             
                                         CO 2 (dissolved)      H 2 CO 3    H   HCO 3
                           • CO 2 (gas)                                   
                                                             carbonicanhy drase  spontaneous
                                                                      Biochemistry: NOTES & OBJECTIVES (page 110 of 165)



                                                         
  - HCO3- buffer:          • pH  6.1 log          [HCO 3 ]
                                             (0.03mM/mm Hg)  (pCO 2 )
   - simplifying assumptions
      - at pH 7.4, all H2CO3 dissociates to HCO3
      - at pH 7.4, all CO2 is dissolved
      - [CO2(d)] = (0.03 mM/mm Hg)·( pCO 2 )
      - open system: CO2 term can be held constant by explusion from the equilibrium
- renal controls: kidney
   - bicarbonate reabsorption
      - Na+ brought in, H+ excreted to maintain electroneutrality
      - H+ in lumen combines with HCO3- in lumen to make H2CO3
      - carbonic anhydrase: breaks down H2CO3 into H2O + CO2
      - CO2 comes into tubular cell, combines with H 2O to make H2CO3 (carbonic anhydrase)
      - H2CO3 breaks down into H+, HCO3-
      - Na+/K+-ATPase: Na+ actively pumped into blood, HCO3 follows to maintain electroneutrality
      - net reaction: HCO3- + Na+ (lumen)  HCO3- + Na+ (blood)
   - net excretion of H+
      - phosphate buffer: major urinary buffer, >50 % (2 Na+, HPO42-)
      - CO2 from blood enters tubular cell, combines with H2O to make H2CO3 (carbonic anhydrase)
      - H2CO3 breaks down into H+, HCO3-
      - H+ excreted via H+ transporting ATPase, Na+ brought in to maintain electroneutrality
      - H+ combines with HPO42- to make H2PO4-; Na+HPO4- is excreted
      - Na+/K+-ATPase: Na+ actively pumped into blood, HCO3 follows to maintain electroneutrality
      - net reaction: CO2 (blood)  HCO3- + Na+ (blood)
- ventilation controls: lung
   - sensors
      - respiratory center of the brainstem: contain both [H +], pCO 2 sensors that moderate rate of ventilation
      - peripheral sensors: similar but smaller effects on rate of ventilation
      - carotid sinus: pCO 2 sensor that can alter ventilation
   - responses
      - alkalosis: hypoventilation to increase alveolar pCO 2 , decrease pH
      - acidosis: hyperventilation to decrease alveolar pCO 2 , increase pH
- chronic acidosis: alteration of nitrogen excretion
   - liver
      - ↑ glutamine synthase:          nitrogen metabolism: local production of amino transporter
      - ↓ carbamoyl-P-synthetase I: nitrogen metabolism: downregulate urea cycle
      - ↓ glutaminase:                 nitrogen metabolism: prefevent futile glutamate/glutamine cycle
   - kidney
      - ↑ glutaminase:                 nitrogen metabolism: local removal of amino group
      - ↑ glutamate dehydrogenase: nitrogen metabolism: reproduction of amino transporter
      - ↑ PEP carboxykinase:           gluconeogenesis
- acid-base disturbances
   - type
      - acidosis: disturbance leading to an increase in [H+] of blood, where pH < 7.4 if not compensated
      - alkalosis: disturbance leading to a decrease in [H+] of blood, where pH > 7.4 if not compensated
   - cause
      - respiratory: disturbance caused by a change in plasma pCO 2
      - metabolic: disturbance caused by a change in plasma [HCO3-]
   - compensation
      - compensation: correction of pH by alteration of the component not changed by the original disturbance
      - kidney may work like hell to take in more HCO3- during metabolic acidosis, but this is not compensation
         - metabolic disturbance  respiratory compensation
         - respiratory disturbance  metabolic compensation
   - anion gap: aid to the diagnosis of a metabolic disturbance
                                                                      Biochemistry: NOTES & OBJECTIVES (page 111 of 165)



    - on average, ([Na+] + [K+]) – ([HCO3-] + [Cl-]) = ~10-20 mEq/L
    - larger anion gap indicates presence of other fixed anions, consistent with a metabolic disturbance
    - anion sources
       - diabetis:          acetoacetate, β-hydroxybutyrate, lactate
       - PKU:               phenylpyruvate, phenyllactate
       - B12 deficiency:    L, D-methylmalonate
       - biotin deficiency: pyruvate, lactate, propionate


Notes: Lecture and Reading

RENAL BIOCHEMISTRY AND FUNCTION IN WATER, ELECTROLYTE, AND H+ BALANCE
electrolyte composition of the intracellular and extracellular fluids
- water
   - constituents of the body
      - water:                 55-60%
      - protein:               15-20%
      - fat:                   15-20%
      - inorganic:             7%
      - carbohydrate:          <1%
   - constituents of water
      - intracellular:         60%
      - interstitial, lymph: 20%
      - dense CT, bone:        15%
      - plasma:                7.5%
   - balance
      - intake: liquids imbibed, water ingested in foods, water produced during metabolism
      - loss: skin, lungs, GI tract, kidneys
- electrolyte concentrations
   - major cations and anions
      - intracellular:         K+, Mg2+            organic phosphates, proteins
      - extracellular:         Na+                 Cl-, HCO3-
   - electroneutrality
      - total anions and cations in each compartment balance each other out
      - membrane potential: charge imbalance is trivial compared to the total ion content
maintenance of water, electrolyte, and H+ balance
- fluid balance
   - intracellular: cellular plasma membrane pumps (somewhat limited)
   - extracellular: kidney (extensive control of total volume, ion composition
- nephron function
   - glomerular filtrate
      - filtration rate: 125 mL/min (180 L/day)
      - composition: same as blood plasma, but with proteins filtered out
   - reabsorption:             Na+, Cl-            ≥ 99.5 %
                               HCO3-               99.9%
                               K+                  93%
                               H2O                 > 99%
   - proximal transport
                               Na+:      actively reabsorbed, primarily in the proximal tubules
                               Cl-:      passively reabsorbed following Na+ (maintain electroneutrality)
                               water: passively reabsorbed following ions
                               others: reabsorbed in the proximal segments
      - cystinuria: relatively common inherited metabolic defect in cysteine transporter
   - distal transport
                               Na+:      actively reabsorbed (aldosterone: absorption)
                               water: passively reabsorbed (antidiuretic hormone: permeability)
                                                                        Biochemistry: NOTES & OBJECTIVES (page 112 of 165)



  - final urine
     - volume:                 0.6 – 1.2 L/day
     - osmolarity:             3-4X plasma osmolarity
     - composition:            urea:                      major solute
                               NH4+, creatine, uric acid: additional secretion of nitrogen
                               sulfate:                   protein catabolism
                               phosphate:                 nucleic acid catabolism
                               organic bases:             ketone bodies, lactate
                               K+:                        concentration usually higher in urine
                               Na+, Cl-:                  concentration usually lower in urine
      - pH:                    much lower than plasma
- nitrogen excretion and creatine clearance
   - nitrogen excretion in the average diet:
                               urea:             85 %
                               creatine:         4-5 %
                               NH4+:             2.5-3 %
                               uric acid:        1.5-2 %
                               others:           5%
   - creatine
      - daily excretion is relatively constant, varying only with muscle mass
      - not reabsorbed from glomerular filtrate
      - creatine clearance: can be used to index the glomerular filtration rate
- differences in metabolism in the renal cortex and renal medulla
   - major consumer of energy: 360 kcal/day
   - cortex: aerobic transport
      - highly vascular, with blood providing necessary substrates for glucose transport
      - metabolism uses glucose, fatty acids, and glutamine
   - medulla: anaerobic transport
      - less vascular, with poorer blood supply and resultantly poorer oxygen supply
      - much of necessary glucose used may be produced by gluconeogenesis in the cortex
ACID BASE BALANCE
buffers
- Henderson-Hasselbach relationships
                                                     
                            • HA  H  A
                                   a  K



                            • pH  pK a     log
                                                 A  


                                                   HA 
- buffering power of the body
   - changes in the [H+] of the blood reflect changes of the [H+] in the body
      - intracellular and extracellular pH is not identical, but a rise/fall in one will be reflected in the other
      - because of this, body buffers become important
   - buffering components in the blood at pH = 7.4
      - hemoglobin, plasma proteins:             42 % of total buffering capacity
      - HCO3- / CO2 system:                      53 % of total buffering capacity
      - phosphate buffer:                        2 % of total buffering capacity
- bicarbonate-CO2 buffer
   - equilibrium:             • CO 2 (gas)   CO 2 (dissolved)      H 2 CO 3    H   HCO 3
                                                                                       
                                                                         carbonicanhy drase          spontaneous   


   - assumptions
      - at pH 7.4, almost all H2CO3 dissociates to HCO3 (actual ratio: CO2 (dissolved):H2CO3 = 800:1)
      - at pH 7.4, almost all CO2 is dissolved
      - [CO2(d)] = (0.03 mM/mm Hg)·( pCO 2 )
                                                          
  - bicarbonate buffer:     • pH  6.1 log          [HCO 3 ]
                                              (0.03mM/mm Hg)  (pCO 2 )
CO2 production
                                                                       Biochemistry: NOTES & OBJECTIVES (page 113 of 165)



- production due to metabolism: 15-20 mol/day
   - determined by metabolic rate, carbon source used in metabolism
   - must be balanced by the rate of alveolar ventilation
- balanced respiratory exchange of CO2: does not result in net change in H+ content
   - rate of ventilation is finely regulated
   - any change in pCO 2 will alter plasma pH
metabolic factors affecting H+ input
- diet: food and means of metabolism can affect H+ content of the body
   - food catabolism
      - acidic vinegar:        neutral:        complete combustion requires no net H+ change
      - acetate salts:         alkalinizing:   complete combustion requires proton consumption
      - citrate salts:         alkalinizing:   complete combustion requires proton consumption
   - protein catabolism
      - carboxylates:          alkalinizing:   complete combustion requires proton consumption
                                               2 R - COO -  H  
                                                                  CO 2  RH
      - amino groups:          acidifying:     complete combustion requires proton production
                                                       
                                                          2R  urea  2H 
                                              2 R - NH 3 
- incomplete metabolism: less efficient metabolism can give an acidifying effect
   - anaerobic respiration: glucose  2 lactate + 2 H+
                              [lactate] may approach 5 mM during heavy work, causing a fall in pH
   - ketone bodies:           palmitoyl residue  4 acetoacetate - + 4 H+
                              in diabetics, total acid load may approach 1 mol/day
renal reabsorption of HCO3- and excretion of H+
- acid base balance: kidney functions in regulation of [H +] in the blood
   - regulated reabsorption of HCO3
   - regulated excretion of H+
- reabsorption of bicarbonate as CO2: the HCO3-/CO2 cycle
   - Na+ brought in, H+ excreted to maintain electroneutrality
   - H+ in lumen combines with HCO3- in lumen to make H2CO3
   - carbonic anhydrase: breaks down H2CO3 into H2O + CO2
   - CO2 comes into tubular cell, combines with H 2O to make H2CO3 (carbonic anhydrase)
   - H2CO3 breaks down into H+, HCO3-
   - Na+/K+-ATPase: Na+ actively pumped into blood, HCO3 follows to maintain electroneutrality
   - net reaction: HCO3- + Na+ (lumen)  HCO3- + Na+ (blood)
- net excretion of H+
   - phosphate buffer: major urinary buffer, >50 % (2 Na+, HPO42-)
   - CO2 from blood enters tubular cell, combines with H2O to make H2CO3 (carbonic anhydrase)
   - H2CO3 breaks down into H+, HCO3-
   - H+ excreted via H+ transporting ATPase, Na+ brought in to maintain electroneutrality
   - H+ combines with HPO42- to make H2PO4-; Na+HPO4- is excreted
   - Na+/K+-ATPase: Na+ actively pumped into blood, HCO3 follows to maintain electroneutrality
   - net reaction: CO2 (lumen)  HCO3- + Na+ (blood)
- metabolic example: lactate production
   - glycogen is broken down into lactate, H+
   - H+ combines with HCO3- to produce CO2 in the blood, which enters the kidney
   - the proton is given off and excreted into the urine, regenerating bicarbonate, which reenters the blood
- excretion of NH3 as NH4+ in chronic acidosis
   - ammonia is highly toxic, and must be excreted to avoid neurological problems
   - for each NH3 excreted as urea, 1 H+ must be independently excreted
      - cost of ammonia detoxificaiton is proton production
      - during prolonged acidosis, liver and kidney adapt to secrete less urea, more NH4+
      - this spares urinary buffers to handle other sources of acid
   - glutamate/glutamine cycle
      - glutamine synthetase:           Glu  Gln          [NH3 ; ATP  ADP + Pi]
      - glutaminase:                    Gln  Glu          [H2O  NH3]
                                                                    Biochemistry: NOTES & OBJECTIVES (page 114 of 165)



      - cycle is central to metabolism and ammonia transport
   - ammonia transport
      - liver: glutamine synthase used to make glutamine, which can safely transport ammonia through the blood
      - kidney: glutaminase used to hydrolyze glutamine into glutamate
         - ammonia: excreted into lumen, drawing a proton with it
         - glutamate: used in gluconeogenesis
   - adaptations: prolonged acidosis
      - liver
         - glutamine synthase:          induced           glutamine synthesis
         - carbamoyl-P-synthetase I: inhibited            urea cycle
         - glutaminase:                 inhibited         glutamine metabolism
      - kidney
         - glutaminase:                 induced           glutamine metabolism
         - glutamate dehydrogenase: induced               gluconeogenesis
         - PEP carboxykinase:           induced           gluconeogenesis
respiratory regulation of acid-base balance
- pH of the blood
   - depends on ratio of [HCO3-]/[CO2(d)]
   - pH can be altered by changing amount of dissolved CO2, as the lung does with degree of ventilation
- hypoventilation: slow, shallow breathing; decreases pH
   - slow breathing causes increase in alveolar pCO 2
   - diffusion of CO2 from blood will be slowed, so pCO 2 of the blood will increase
- hyperventilation: rapid, deep breathing
   - rapid breathing causes decrease in alveolar pCO 2
   - diffusion of CO2 from blood will be increased, so pCO 2 of the blood will decrease
- control of ventilation by the lung
   - respiratory center of the brainstem: contain both [H +], pCO 2 sensors that moderate rate of ventilation
   - peripheral sensors: similar but smaller effects on rate of ventilation
   - carotid sinus: pCO 2 sensor that can alter ventilation
metabolic and respiratory disturbances of acid-base balance
- classification
   - reasoning
      - HCO3- / CO2 are the major blood buffer
      - blood is a clinically accessible fluid
      - when H+ is produced, HCO3- is lost (H+ + HCO3-  CO2 exhaled) unless regenerated from CO 2 in the kidney
   - definitions
      - disturbances: type
         - acidosis: disturbance leading to an increase in [H+] of blood, where pH < 7.4 if not compensated
         - alkalosis: disturbance leading to a decrease in [H+] of blood, where pH > 7.4 if not compensated
      - disturbances: cause
         - respiratory: disturbance caused by a change in plasma pCO 2
         - metabolic: disturbance caused by a change in plasma [HCO3-]
      - compensation
         - compensation: correction of pH by alteration of the component not changed by the original disturbance
         - kidney may work like hell to take in more HCO3- during metabolic acidosis, but this is not compensation
            - metabolic disturbance  respiratory compensation
            - respiratory disturbance  metabolic compensation
- metabolic acidosis
   - metabolic acidosis: most common acid-base disturbance seen by a physician
   - diagnosis: acidosis caused by decrease in plasma [HCO3-]
   - examples
      - diabetis: incomplete metabolism causes increased H+ input
      - phenylketonuria: incomplete metabolism causes increased H+ input
      - kidney malfunction: defect causes decreased H+ output
                                                                       Biochemistry: NOTES & OBJECTIVES (page 115 of 165)



  - compensation: increased ventilation causing decreased pCO 2 (limit of ~10 mm Hg)
  - additional diagnostic features: anion gap
     - blood ions can be cheaply determined
     - on average, ([Na+] + [K+]) – ([HCO3-] + [Cl-]) = ~10-20 mEq/L
     - larger anion gap indicates presence of other fixed anions, consistent with metabolic disturbance
     - anion sources
        - PKU:     phenylpyruvate, phenyllactate
        - B12:     L, D-methylmalonate
        - biotin: pyruvate, lactate, propionate




51. The Feed/Fast Cycle – Integration of Protein Metabolism and
        H+ Balance
Study Guide
know the following:
- protein catabolism (and urea synthesis) is relatively invariant in the fed and short-term fasted states
   - fed state: excess protein is typically present at a level approaching 75 g / day
   - fasted state: muscle proteins broken down for gluconeogenesis at a level of about 75 g/day
- short term fasting
   - protein resynthesis: slows due to lack of insulin
   - protein turnover: sufficient to provide gluconeogenic substrates
   - preferential release of amino acids
      - muscle:     preferentially releases Ala, Ser, and Gln (often made from Ile, Val, and Leu)
      - liver:      preferentially takes in Ala and Ser; takes in Gln during early fasting for gluconeogenesis
      - kidney:     takes in Gln during later fasting for gluconeogenesis
- long-term (> 1 week) fasting: metabolic changes
   - ketone bodies: increase in blood, causing chronic acidosis
   - brain
      - ketone bodies: adapts to use ketone bodies, thus requiring less glucose
      - glucose: adapts to use some glucose anaerobically, synthesizing lactate (gluconeogenic substrate)
   - liver, kidney
      - nitrogen excretion: adapt to excrete less nitrogen as urea, more as NH 4+
      - gluconeogenesis: kidney adapts to becoming the major gluconeogenic organ
      - purpose: decreases load on other urinary buffers, facilitating H+ excretion and minimization of acidosis
   - muscle
      - protein breakdown: with glucose needs of the brain minimized, break down less protein
      - purpose: prolonges time one can fast


Notes: Lecture and Reading
protein metabolism during feeding
- recommended distribution: 70 kg human, 3000 kcal diet
   TABLE: Recommended Diet for 70 kg Active Man on a 3000 kcal Diet
                        type                             % of diet         calories in diet
     carbohydrate       80% complex                      55-60%            1700 kcal
     fat                2/3 mono, polyunsaturated        30%               900 kcal
     protein            high quality                     12-15%            400 kcal
   - note that the protein exceeds the minimal standard set by RDA for normal protein resynthesis
- total metabolic rate
   - basal metabolic rate:            60 % of total metabolic rate (~1800-2000 kcal for 70 kg man)
   - thermal effect of food:          5-10 % of total metabolic rate
                                                                        Biochemistry: NOTES & OBJECTIVES (page 116 of 165)



   - physical activity                     ~30 % of total metabolic rate
- variations
   - men (vs. women, children): higher basal metabolic rate
   - thermal effect: energy expended in digestion, stimulation of metabolism
   - physical activity: varies 10-fold between laziest and most active
- American diet
   - more fat than recommended: 42 % of caloric intake
   - less CHO than recommended: 46 % of caloric intake
   - correct protein:                      12% of caloric intake
- protein catabolism (and urea synthesis) is relatively invariant in the fed and short-term fasted states
   - liver: relatively high levels of certain enzymes
      - alanine aminotransferase           (ALA) alanine metabolism (high in liver)
      - aspartate aminotransferase (AST) urea metabolism
   - during fasting states, enzyme levels do not change
      - rate of protein breakdown: 75 g/day, close to the rate of excess amino acid catabolism in American diet
      - in either state, liver will still metabolize alanine, still produce urea
protein metabolism during fasting and adaptations to a long term fast
- short term fasting
   - gluconeogenesis from protein: no new control needed
      - protein turnover in muscle is ~100 g/day
      - during short term fasting, muscle exports ~75 g protein per day to the liver for gluconeogenesis
      - because normal protein resynthesis has stopped, this protein simply comes from normal degradation
   - preferential release of amino acids
      - muscle:       preferentially releases Ala, Ser, and Gln (often made from Ile, Val, and Leu)
      - liver:        preferentially takes in Ala and Ser; takes in Gln during early fasting for gluconeogenesis
      - kidney:       takes in Gln during later fasting for gluconeogenesis
- long-term (> 1 week) fasting: metabolic changes
   - brain adapts to utilize ketone bodies, muscle and heart stop using ketone bodies to spare for the brain
      - lack of muscle, heart utilization: leads to ketosis, ketonuria
      - brain use of ketone bodies: minimizes need for glucose
   - brain partially shifts from anaerobic to aerobic glycolysis; lactate used in gluconeogenesis
   - lower glucose requirements of the CNS lead to decreased muscle protein breakdown
   - kidney supplements liver as major site of gluconeogenesis, alters nitrogen excretion to minimize acidosis
      - liver
         - ↑ glutamine synthase:                   nitrogen metabolism: local production of amino transporter
         - ↓ carbamoyl-P-synthetase I:             nitrogen metabolism: downregulate urea cycle
         - ↓ glutaminase:                          nitrogen metabolism: prefevent futile glutamate/glutamine cycle
      - kidney
         - ↑ glutaminase:                          nitrogen metabolism: local removal of amino group
         - ↑ glutamate dehydrogenase:              nitrogen metabolism: reproduction of amino transporter
         - ↑ PEP carboxykinase:                    gluconeogenesis




52. & 53. General Principles of Hormone Action
Study Guide
- hormone receptor interaction
   - Kd: dissociation constant, describing the concentration to which half of the receptors are dissociated
      - high number: low affinity
      - low number: high affinity (usually described as Kd ≤ 1 x 10-9)
   - regulation
      - modulate hormone levels (turnover, binding properties)
      - modulate receptor levels (number, binding properties)
                                                                      Biochemistry: NOTES & OBJECTIVES (page 117 of 165)



   - location
      - water-soluble hormone receptors: cell surface
      - lipophilic hormone receptors: intracellular, often within the nucleus
   - % receptor occupancy
      - hormonal effects are dependent on both hormone concentration and on % of receptor occupancy
         - some physiological effects are stimulated by very low levels of receptor occupancy
         - others require much greater levels
      - this allows differentiation based on binding properties (e.g. with insulin)
- mechanisms of signal transduction
   - water-soluble hormones: extracellular stimulation via second messenger
      - hormone binds receptor on cell surface, activating a second messenger
      - second messenger brings about intracellular effects, producing the physiological response
   - lipophilic hormones: intracellular stimulation via second messenger or (more commonly) altered transcription
      - hormone binds receptor on cell surface, forming an H-R complex
      - H-R complex brings about intracellular effects, producing the physiological response
- second messengers: mechanisms
   - cAMP
      - mechanisms of increasing cAMP: adenylate cyclase complexes
         - hormone binds extracellular receptor, causing the α-GDP subunit to lose GDP, gain GTP, dissociate
            - Gsα: activates adenylate cyclase
            - Giα: inhibits adenylate cyclase
            - Gqα: activates phospholipase C
         - intrinsic α-GTPase terminates the signal, allowing the receptor-(α, β, γ) complex to reform
      - protein phosphorylation
         - cAMP binds regulatory proteins, releasing active kinases (PKA) that phosphorylate cellular enzymes
         - signal is terminated via degradation of cAMP, constitutive phosphatase activity
      - transcriptional control: cAMP-regulatory element binding protein (CREB)
         - PKA causes phosphrylation, activation of CREB, which binds a responsive gene, upregulates transcription
         - signal is terminated via constitutive phosphatase activity
   - intracellular Ca2+
      - mechanisms of increasing Ca2+
         - release of sequestered Ca2+ contained largely within the ER
         - influx of extracellular Ca2+ (typical extracellular concentration: 1-10 μm)
      - intracellular Ca2+ acts as a second messenger in several cell processes
         - direct Ca2+-dependent activation of protein kinases, phospholipases
         - indirect activation of protein kinases via binding of calmodulin
   - phospholipids: used in cellular responses that mobilize Ca2+
      - PI metabolized to PIP, then to PIP 2 by kinases
      - Gq-activated phospholipase C (PI-PLC) metabolizes PIP2 to DAG, IP3
         - DAG: activates PKC isoforms
         - IP3: mobilizes Ca2+ from intracellular stores, such as the ER
- hormonal regulation
   - mechanisms
      - simple classical: controlled variable inhibits release of the hormone from the gland that releases it
      - complex-hormonogen: signal activates a hormongen, causing a physiological change that cancels the signal
      - hypothalamic-pituitary: hypothalamic signal causes release of an RH, which acts on the anterior pituitary to
                 release a tropic hormone, which causes a physiological change that feeds back on the hypothalamus
   - other factors
      - transport: hydrophobic hormones require passage on carrier proteins, while water-soluble are often free
      - metabolism: hormone can be given an altered or different activity; often done in the liver
      - permissive effect: the prior action of one hormone allows another hormone to exert its effects


Notes: Lecture and Reading

ENDOCRINE SYSTEM
                                                                       Biochemistry: NOTES & OBJECTIVES (page 118 of 165)



- endocrine system: hormone-producing endocrine glands of the body
- endocrine glands: ductless glands that secrete products directly into the bloodstream
general hormone properties
- hormonal communication
   - endocrine: action on distant target tissue through use of the bloodstream
   - paracrine: action on nearby target tissue through diffusion to neighboring cells
   - autocrine: action on the cell producing the hormone
- hormone diversity and specificity
   - mechanism: endocrine gland  hormone in blood, interstitium  target tissue  change in variable(s)
   - target tissue: site of action of a hormone
   - end-organ response: change in the controlled variable
- hormone receptors
   - equilibrium expression:             [H] + [R]  [HR]
                                                 [HR] (reflection of hormone binding)
      - association constant Ka:          Ka 
                                                [H][R]
                                                [H][R] (reflection of ease with which hormone dissociates)
      - dissociation constant Kd:         Kd 
                                                 [HR]
   - characteristics of hormone receptor interactions
      - complex formation: dependent on both H and R
      - high affinity: typically, Kd ≤ 1 x 10-9 (higher number means lower affinity)
      - saturable: small receptor number / cell, limiting the level of signal produced and preventing overexpression
      - regulation: can increase or decrease either hormone or receptor, or modulate receptor affinity (K d)
      - affinity: can be influenced by hormone binding and other factors, regulating cellular responsiveness
      - turnover: can be influenced by hormone binding and other factors, regulating cellular responsiveness
      - occupancy: some end responses only occur with varying levels of receptor occupancy
      - multiple receptor types: allows selective response to different levels, tissues, or regulation pathways
- general chemical classes
   - amino acid derivatives:             epinephrine, thyroxine
   - peptides:                           hypothalamic releasing hormones
   - polypeptides:                       insulin, glucagon
   - steroid hormones:                   sex hormones, adrenal cortical hormones
   - phospholipid derivatives:           platelet-activating factor
   - lipid-soluble vitamins:             retinoids, vitamin D
   - adenine derivatives:                ATP, ADP, adenosine
   - gases:                              nitric oxide
- time course of action
   - speed of action: ranges from seconds (adrenaline) to days (thyroxine)
   - duration (half lives): ranges from seconds to days; generally have half lives of < 1 hour
- effective concentrations
   - general range: 10-12 to 10-9 M
   - reason: low level of concentration is a consequence of the high affinity of most HR binding
- hormonogens (pro hormones)
   - hormonogen: circulating precursor hormones that are activated to true hormones
   - localization: can occur at various body sites, including the target tissue
mode of action of hormones
- water-soluble hormones: cannot easily pass through membrane
   - mode of action: production of a second messenger
      - hormone binds receptor (usually an IMP) on the extracellular surface
      - signal is transmitted to the rest of the cell via an intracellular second messenger
      - second messenger produces an altered cellular response
      - altered cellular response produces altered physiological response
   - time course: often quite rapid
   - internalization
      - internalization: receptor-mediated endocytosis of the HR complex
      - purpose: receptor down-regulation during continual exposure, receptor recycling, and signalling
                                                                     Biochemistry: NOTES & OBJECTIVES (page 119 of 165)



- lipid-soluble hormones: readily pass through membrane
   - mode of action: altered transcription
      - hormone passes through membrane, binds receptor within the cell
      - HR complex binds DNA or DNA-protein complexes (specific interaction)
      - binding alters cellular transcription
      - altered cellular response produces altered physiological response
   - mode of action: production of a second messenger (e.g. Ca 2+ flux)
      - hormone passes through membrane, binds receptor within the cell to produce a second messenger
      - second messenger produces an altered cellular response
      - altered cellular response produces altered physiological response
   - time course: minutes (Ca2+ flux) to days (altered transcription)

- general hormone responses: targets
   - transporters:       alteration of transport through the cell membrane
   - enzymes:            modulation of enzyme activity
   - proteins:           alteration of transcriptional, translational actions on protein synthesis
   - small molecules:    modulation of synthesis, flux of small molecules which then act as second messengers

INTRACELLULAR MESSENGERS: OVERVIEW
cAMP
- overview of cAMP action
     tissue                hormone causing increase in [cAMP]          cellular response
     adipose               adrenaline (β receptors), glucagon          ↑ lipolysis
     liver, muscle         adrenaline (β receptors)                    ↑ glycogenolysis, ↓ glycogen synthesis
                           glucagon (liver only)                       ↑ gluconeogenesis (liver only)
     ovarian follicle      FSH, LH                                     ↑ estrogen, progesterone synthesis
     adrenal cortex        ACTH                                        ↑ cortisol, aldosterone synthesis
     cardiac muscle        adrenaline (β receptors)                    ↑ rate of contraction
     thyroid               TSH                                         ↑ thyroxine synthesis, secretion
     osteoclasts           parathyroid hormone                         ↑ Ca2+ resorption from bone
   - hormone: first messenger, stimulates target cells to produce cAMP
   - cAMP: second messenger that promotes a change in cellular activity, depending on the cellular enzyme profile
- synthesis and degradation of cAMP
   - ATP  cAMP               [adenylate cyclase:  PPi]
   - cAMP  5’-AMP            [phosphodiesterase: H2O ]
- G protein modulation of cAMP
   - adenylate cyclase complexes
      - localization:         cell membrane
      - composition:          receptor (α,β,γ)
                              G-protein (guanine nucleotide-binding protein)
                              adenylate cyclase isoform
   - mechanism
      - hormone binds receptor, which causes the α-GDP subunit to lose GDP, gain GTP, dissociate
      - α-GTP subunit modulates enzyme activity
         - Gsα: activates adenylate cyclase
         - Giα: inhibits adenylate cyclase
         - Gqα: activates phospholipase C
      - GTP is eventually hydrolyzed to GDP by the intrinsic GTPase of the α subunit
      - α-GDP is formed, terminating the signal
      - α-GDP associates with β, γ, reforming the receptor-(α, β, γ) complex
   - other considerations
      - there are numerous G proteins, many of which are monomers that are not self regulated
      - as such, many H-R systems are coupled to cellular systems that can affect enzymes, transport systems like:
         - Ca2+, K+ specific channels
         - phosphlipases and phosphodiesterases
                                                                       Biochemistry: NOTES & OBJECTIVES (page 120 of 165)



         - numerous protein kinase cascades
- biological role of cAMP: intracellular messenger mediating many hormonal effects
   - a given hormone is recognized by receptors specific to target tissues
   - different cell types express different intracellular enzymes that are regulated by cAMP-dependent processes
- protein phosphorylation mechanism of cAMP action
   - mechanism: R2C2 (inactive) + 4 cAMP  2 R-cAMP2 + 2 C (where C is the active kinase)
   - function:       use in enzyme cascade systems
   - regulation: deactivation via phosphoprotein phosphatases
- transcriptional control by cAMP: cAMP-regulatory element binding protein (CREB)
   - mechanism: 2 CREB  2 CREB-P (protein kinase A: 2 ATP  2 ADP)
                     2 CREB bind CRE on responsive gene, upregulating its transcription
   - example:        phosphoenolpyruvate carboxykinase (PEPCK)
intracellular Ca2+
- overview
   - intracellular Ca2+ typically low (10-100 nM)
   - in response to certain signals, Ca2+ can be increased, thereby acting as a second messenger
   - many enzymes show a Kd for Ca2+ of ~1 μm
- mechanisms of increasing Ca2+
   - release of sequestered Ca2+ contained largely within the ER
   - influx of extracellular Ca2+ (typical extracellular concentration: 1-10 μm)
- hormonal effects which can be mediated by Ca2+ modulation
   - hepatic glycogen degradation, gluconeogenesis: adrenaline (α1 receptors):
   - hormone secretory processes:                          insulin release from pancreatic β cells, among others
   - muscle contraction, vasoconstriction:                 oxytocin, adrenaline, angiotensin II
- mechanisms of Ca2+ action
   - direct activation of Ca2+-dependent protein kinases, phospholipases
   - calmodulin: Ca2+ binding protein that, upon activation, binds specific proteins to regulate activity
      - Ca2+-calmodulin-activated protein kinases
      - phosphorylase kinase
      - phospholipase A2 (certain forms)
phospholipids
- the role of phospholipids in hormone action
   - phospholipid metabolites: can act as intracellular, intercellular signaling molecules, activating phospholipases
   - phospholipases
      - PLA1: hydrolyzes ester bond of fatty acid at C1
      - PLA2: hydrolyzes ester bond of fatty acid at C2
      - PLC: hydrolyzes phosphodiester bond between glycerol, phosphate of head group
      - PLD: hydrolyzes phosphodiester bond between phosphate and head group
- phospholipase regulatory molecules
   - isoforms of PLA2:        Ca2+, Ca2+-calmodulin, phosphorylation, G-protein interaction
   - isoforms of PLC:         heterotrimeric Gq, PLC-β, tyrosine phosphorylation (by PLC-γ1, PLC-γ2)
   - isoforms of PLD:         small MW G proteins
- PLA2 activation
   - many hormone systems that activate Ca2+ mobilization result in enhanced PLA2 activity
      - PLA2: linked to hormone-stimulated release of arachidonic acid
      - arachidonic acid: precursor for eicosanoids
      - eicosanids: FA derivatives that include prostaglandins, thromboxanes, leukotrienes
   - eicosanids: mediate numerous processes including inflammation, smooth muscle contraction, clotting
   - non-steroidal anti-inflammatory drugs (NSAIDs): inhibit production of eicosanoids
- phosphoinositide hydrolysis in hormone signal transduction
   - phosphatidylinositol (PI): acts as a mediator in the cellular response of hormones that mobilize Ca 2+
   - process
      - phosphatidylinositol (PI)  phosphatidylinositol-phosphate (PIP) [PI-4-kinase]
      - phosphatidylinositol-phosphate (PIP)  phosphatidylinositol 4,5-bisphosphate (PIP2) [PI-5-kinase]
      - PIP2  diacylglycerol (DAG) + 1,4,5-trisphosphoinositol (IP3) [PI-PLC]
   - product functions
                                                                       Biochemistry: NOTES & OBJECTIVES (page 121 of 165)



      - DAG: activates isoforms of protein kinase C
      - IP3: mobilizes Ca2+ from intracellular stores
   - regulation: PI-PLC
      - PI-PLC: phosphoinositide-specific phospholipase C
      - Gq: G protein complex that regulates activity of PI-PLC
   - summary
      - hormonal signal causes activation of Gq protein complex
      - this activates PI-PLC, which hydrolyzes PIP2 to DAG and IP3
      - DAG activates PKC, IP3 mobilizes Ca2+ from intracellular stores
metabolic regulation and homeostasis
- role of hormones in regulating homeostasis – feedback controls
   - overview
      - homeostasis: maintained through combined efforts of endocrine, neural systems
      - feedback control: level of one substance attenuates production or release of another substance or hormone
   - feedback systems
      - simple classical type
         - mechanism:         controlled variable inhibits release from the gland that causes its release
         - example:           insulin-glucose, parathyroid-hormone-Ca2+ levels
      - complex-hormonogen type
         - mechanism:         signal activates hormonogen, which effects a physiological change to cancel the signal
         - example:           angiotensinogen-aldosterone
      - hypothalamic-pituitary-target endocrine organ system
         - mechanism:         hypothalamic signal secretes a releasing hormone, targeting anterior pituitary
                              anterior pituitary releases a tropic hormone, targeting tissue
                              tropic hormone feeds back on hypothalamus or anterior pituitary
         - example:           CRF-ACTH-cortisol
- other factors influencing hormone levels
   - transport of hormones
      - hydrophobic hormones:          typically carried on plasma carrier proteins
      - water-soluble hormones:        often present as free hormone, though carriers may facilitate/regulate as well
      - albumin: general universal carrier of hormones
   - metabolism of hormones
      - modulation:           metabolism resulting in more, less, or inactive products, thus altering the response
      - conversion:           metabolism resulting in hormones with different hormonal activity
      - localization:         degradation typically in the liver, though other tissues can process as well
   - insufficient response
      - extent of a hormonal response can be limited by the physical capacity of a gland to secrete a hormone
      - example: with high amounts of adipose tissue, pancreas cannot make enough insulin to meet demand
- interdependence of hormones and “permissive effects”
   - hormonal response can be dependent on multiple signals with opposing effects
      - decrease blood glucose:        insulin
      - increase blood glucose:        glucagon, adrenaline
   - permissive effect: the prior action of one hormone allows another hormone to exert its effects
      - estrogen:             induces expression of progesterone receptor in the uterus
      - progesterone:         only able to exert uterine effects with sufficient receptors

TABLE: Major Endocrine Glands Producing Polypeptide or AA-derived Hormones
 endocrine gland   hormone                   principal target            principal effect
 adenohypophysis   prolactin                 mammary gland               proliferation, milk formation
                   ACTH                      adrenal cortex              synthesis, secretion of adrenal cortical steroids
                   thyrotropin (TSH)         thyroid                     synthesis, secretion of thyroxine and triiodothyronine
                   somatotropin (GH)         general                     growth of bone and muscle
                                                                         anabolic effect on Ca2+, phosphate, nitrogen metabolism
                                                                         metabolism of CHO and lipid
                                                                         elevation of muscle and cardiac glycogen
                   lutenizing hormone (LH)   ovary                       lutenization
                                                                         progesterone secretion
                                                                                    Biochemistry: NOTES & OBJECTIVES (page 122 of 165)



                                                 testis                               development of interstitial tissue
                                                                                      secretion of testosterone
                   follicle-stimulating          ovary                                follicular development
                   hormone (FSH)                                                      with LH, secretion of estrogen and ovulation

                                                 testis                               development of seminiferous tubules
                                                                                      early stages of spermatogenesis
                   α- and β-lipotropins          adipose cells                        release of lipid
 neurohypophysis   oxytocin                      smooth muscle (esp. uterine)         contraction, parturition

                                                 mammary gland, postpartum            ejection of milk
                   vasopressin (ADH)             kidney tubules                       water reabsorption

                                                 arterioles                           increase blood pressure
 pancreas          insulin                       general                              utilization of CHO
                                                                                      increase CHO stores, stimulate fat and protein synthesis

                                                 adipose tissue                       lipogenesis
                   glucagon                      liver                                glycogenolysis, gluconeogenesis

                                                 adipose tissue                       lipolysis (release of lipid)
                   somatostatin                  pancreas, adenohypophysis            inhibit secretion of insulin, glucagon, and somatotropin
 adrenal medulla   epinephrine,                  liver and muscle                     glycogenolysis, gluconeogenesis (liver)
                   norepinephrine
                                                 adipose                              release of lipid
 thyroid           thyroxine, triiodothyronine   general                              metabolic rate, O2 consumption of tissues
                                                                                      general growth and metabolic activities

                   calcitonin                    skeleton                             Ca2+ metabolism
 parathyroids      parathormone                  skeleton, kidney, GI tract           Ca2+, phosphate metabolism
 atria of heart    ANF                           kidney, vascular system, adrenal     Na+ excretion, blood volume, BP
                                                 cortex

TABLE: Major Endocrine Glands Producing Steroid Hormones
 endocrine gland   hormone                       principal target                    principal effect
 testis            testosterone                  male accessory sex                  maturation, normal function

                                                 general                             development of male secondary sex characteristics
 seminal vesicle   prostaglandins                smooth muscle, adipose,             blood pressure
                                                 platelets, brain, GI tract, etc.    smooth muscle contraction
                                                                                     lipid metabolism
                                                                                     platelet aggregation
                                                                                     excitability
 ovary             estrone, estradiol            female accessory sex organs         maturation, normal cyclic function

                                                 mammary glands                      development of duct system

                                                 general                             development of secondary female sex characteristics
 corpus luteum     progesterone                  uterus                              preparation for ovum implantation
                                                                                     maintenance of pregnancy

                                                 mammary glands                      development of alveolar system
 adrenal cortex    adrenal cortical steroids     general

                   aldosterone                                                       electrolyte metabolism

                   corticosterone, cortisol                                          metabolism of proteins, CHO, and lipids
                                                                                     maintenance of circulatory, vascular homeostasis
                                                                                     inflammation, immunity, resistance to infection

SUPPLEMENTARY: overview of hormone systems not to be discussed
- alimentary hormones: GI tract polypeptide hormones secreted into the blood, regulating digestion
- relaxin: protein hormone of the placenta, corpus luteum, and uterus that increases cervical distensibility
- kinins: oligopeptides formed from circular precursors that affect smooth muscle contractions of hollow organs
- erythropoietin: glycoprotein produced from circulating precursors, stimulating production of RBCs
- endorphins, enkephalins: oligopeptides produced in the CNS from circulation, having opiate-like actions
                                                                        Biochemistry: NOTES & OBJECTIVES (page 123 of 165)



- leptin: adipose peptide hormone serving as a sensor of energy stores, regulating appetite in the CNS
- growth factors and cytokines: general properties
   - most are polypeptides
   - effects initiated on external surface of cells via receptor-ligand complexes
   - GF-R complex gives a specific growth response
   - factor, receptor removed via receptor-mediated endocytosis




54. Pancreatic Hormones – Insulin
Study Guide
insulin
- insulin chemistry
   - structure: two polypeptide chains joined by two interchain disulfide bridges, with one intrachain bridge on A
   - synthesis: produced in pancreatic β cells
      - synthesis: proinsulin synthesized on ribosomes of RER of β cells
      - transport: proinsluin transferred to Golgi, packaged into granules
      - cleavage: proinsulin cleaved into insulin (90-95% efficiency)
      - storage:      insulin is stored in granules as a Zn2+/insulin complex along with C-peptide
      - secretion: upon proper signals (primarily high blood glucose), granules are secreted into the blood
- regulation
   - process
      - mechanism: ATP-dependent, Ca2+-dependent; cAMP modulates levels, but is not required
      - degradation: promoted by thyroid hormones (T 3, T4)
   - factors
      - blood glucose concentration: primary means of insulin modulation
      - method of glucose intake: intravenous vs. oral
         - intravenous: glucose gives a biphasic response at physiological concentrations; less potent than oral
         - oral: insulin levels arise after glucose contacts the duodenum, before blood glucose has risen (anticipatory)
      - amino acids (Arg, Leu): increase insulin secretion (also increases glucagon; does not significantly alter I/G)
      - adrenaline: lowers insulin release via α2 (Gi-linked) receptor; conserves glucose for non insulin-dependents
      - growth hormone (GH), glucocorticoids: enhance insulin secretion via islet hyperplasia (developmental)
- biological effects
   - lowers blood glucose: stimulates muscle, adipose uptake
   - stimulates glycolysis: induces glucokinase, pyruvate kinase expression
   - inhibits gluconeogenesis: decreases availability of substrates, decreases activity/amount of enzymes
   - inhibits adipose lipolysis: inhibits hormone-sensitive lipase (decreased cAMP, decreased PKA)
   - stimulates liver, adipose lipogenesis: induces lipogenic enzymes (e.g. acetyl-SCoA carboxylase, FA synthase)
   - stimulates general protein synthesis: facilitates, activates amino acids, decreases protein degradation
   - promotes growth: very high levels of insulin
- signal transduction
   - protein tyrosine kinase
      - autophosphorylates tyrosine residues, which leads to phosphorylation of IRS-1, IRS-2
      - phosphorylated IRS-1, IRS-2 serve as docking sites for multiple effector molecules
   - G protein
      - Gi protein activated, downregulating adenylate cyclase, reducing cAMP levels
      - cAMP phosphodiesterase activated, further reducing cAMP levels
- disease states
   - hypoinsulinism (diabetes mellitus): abnormality in the synthesis, secretion, and/or effect of insulin
      - type I: poor insulin production leading to chronic high blood sugar
      - type II: poor receptor response leading to chronic high blood sugar
   - hyperinsulinism: excessive insulin production/overdose, leading to low blood sugar; much more dangerous
                                                                        Biochemistry: NOTES & OBJECTIVES (page 124 of 165)




Notes: Lecture and Reading

TABLE: Overview of Major Pancreatic Hormones
 hormone       site of synthesis   principal site of action          principal phenomena
 insulin       β cells             muscle, liver                     utilization of CHO
                                                                     stimulation of protein synthesis

                                         adipose                     lipogenesis
 glucagon           α cells              liver                       glycogenolysis

                                         adipose                     release of lipid
 somatostatin       δ cells              pancreatic islet cells      inhibition of insulin, glucagon secretion

insulin chemistry
- structure
   - two polypeptide chains (A and B) joined by two interchain disulfide bridges; A intrachain disulfide bridge
   - note: can be inactivated by proteolytic enzymes, and thus cannot be taken orally
- conservation across species
   - animal insulin preparations are active in humans, though there can be antigenic problems
   - porcine insulin: most closely related (substitution of alanine for threonine at B30)
- solubility
   - aggregates in solution
   - complexes with basic proteins (e.g. protamine) and divalent cations (e.g. Zn2+)
   - depot preparations: complexes of low solubility that dissociate slowly, giving a timed release
insulin biosynthesis, storage, and release
- hormones
   - preproinsulin: translation product of insulin gene; cleaved into proinsulin
   - proinsulin: insulin precursor with little insulin-like activity (2%), but partial immunoreactivity (50%) of insulin
   - insulin: final hormone product secreted by the pancreatic β cells
- key points in insulin biosynthesis
   - synthesis:      proinsulin synthesized on ribosomes of RER of β cells
   - transport:      proinsluin transferred to Golgi, packaged into granules
   - cleavage:       proinsulin cleaved into insulin (90-95% efficiency)
   - storage:        insulin is stored in granules as a Zn2+/insulin complex along with C-peptide
   - secretion:      upon proper signals (primarily high blood glucose), granules are secreted into the blood
regulation of insulin release
- overview
   - requirements for insulin release
      - triggering signal                                  (e.g. high blood glucose)
      - energy source for ATP-dependent exocytosis (can be glucose)
   - primary regulation: blood glucose concentrations
   - triggering process: Ca2+-dependent
   - intracellular cAMP
      - high [cAMP] enhances insulin release
      - low [cAMP] decreases insulin release
      - many hormones that affect glucose levels do so by altering cAMP levels in pancreatic β cells
      - note: cAMP is not required for insulin release; it merely modulates the level
- biological half life
   - half life: 5-15 minutes
   - thyroid hormones (T3, T4): promote insulin degradation, decreasing its half life
- factors stimulating or inhibiting insulin secretion
   - blood glucose concentration: primary means of insulin modulation
   - method of glucose intake: intravenous vs. oral
      - intravenous: glucose gives a biphasic response at physiological concentrations
                                                                        Biochemistry: NOTES & OBJECTIVES (page 125 of 165)



         - phase I: rapid discharge from β cell granules, lasting 15-20 minutes before declining
         - phase II: biosynthesis of insulin, peaking between 30-60 minutes
      - oral: insulin levels arise after glucose contacts the duodenum, before blood glucose has risen
         - blood glucose does not mediate the initial rise; instead, it is GI hormones (insulin-releasing polypeptides)
         - anticipatory response: body preparation for a rise in glucose by prematurely releasing insulin
      - comparison: the insulin response to oral glucose is much greater (~250 – 300 % of IV)
   - amino acids (Arg, Leu): increase insulin (and glucagon) secretion
      - mechanism
         - stimulate release of GI factors that enhance insulin secretion from β cells
         - because they also stimulate glucagon release, I/G does not change markedly
      - hormonal effects: logic of using protein to stimulate insulin secretion
         - increased insulin: promotes protein synthesis
         - increased glucagon: promotes gluconeogenesis
      - note: amino acids cannot be stored, so their only other fate would be excretion
   - adrenaline: lowers insulin release via an α2 receptor
      - logic
         - adrenaline lowers insulin to conserve glucose for the brain and exercising muscle (not insulin-dependent)
         - note: insulin-dependent muscular glucose uptake applies only to resting muscle
      - mechanism: binds α2 receptor, lowering intracellular cAMP via G i, thus attenuating insulin release
   - growth hormone (GH) and glucocorticoids: enhance insulin secretion
      - GH: long term developmental hormone
      - mechanism: produce hypertrophy and hyperplasia of the islets, increasing basal and stimulable output
      - insulin resistance: downregulation of insulin receptors caused by chronic exposure to insulin
insulin biological effects
- lowers blood glucose
   - stimulates transport of glucose into muscle, adipose
- stimulates glycolysis
   - induces glucokinase expression
   - induces pyruvate kinase expression, stimulates its activity
- inhibits gluconeogenesis
   - decreases availability of gluconeogenic substrates, such as amino acids and glycerol
   - decreases activity/amount of gluconeogenic enzymes (e.g. inhibition of PEP carboxykinase synthesis)
- inhibits lipolysis in adipose tissue
   - inhibits hormone-sensitive lipase via decreased cAMP (Gi), decreased PKA activity
- stimulates lipogenesis in liver and adipose tissue (from excess CHO)
   - induces lipogenic enzymes (e.g. acetyl-SCoA carboxylase, FA synthase)
- stimulates general protein synthesis in most body tissues
   - facilitates entry of amino acids into cells
   - increases activation of amino acids
   - increases activity of initiation and/or elongation factors required with ribosomes
   - decreases rate of protein degradation in muscle, other tissues
- promotes growth (very high levels of insulin)

TABLE: Signal Responsiveness: Concentrations Required for Different Physiological Effects of Insulin
 physiological action                 [insulin] (μU/mL) clinical implications
 stimulation of glucose uptake        100-300           high [insulin] attained only after meals, and insulin
 into sensitive tissues (e.g. resting                   primarily affects postprandial glucose uptake
 skeletal muscle)
 suppression of glycogenolysis,       10-30             major means by which insulin decreases blood glucose
 gluconeogenesis by liver
 inhibition of protein degradation 5-20                 permits growth, prevents weight loss and tissue
                                                        breakdown, and decreases AA presented to the liver for
                                                        GNG
 inhibition of ketogenesis in liver 5-20                prevents ketosis
 inhibition of lipolysis              5                 prevents ketosis by decreasing FA presented to liver;
                                                                       Biochemistry: NOTES & OBJECTIVES (page 126 of 165)



                                                                prevents weight loss
   - hormonal effects are dependent on hormone concentration and % of receptor occupancy
   - some effects are stimulated by very low levels, while others require much greater hormonal levels
      - insulin: lipolysis is inhibited even at very low concentrations
      - consequently, type I diabetes mellitus typically leads to lipolysis, while type II frequently does not
insulin receptor mode of action
- receptor structure
   - location: cell surface
   - structure: transmembrane glycoprotein (α2β2), with cross links between α-α and α-β pairs
      - α subunit: most responsible for binding specificity
      - β-subunit: insulin-activated protein-tyrosine kinase that traverses the membrane
- mode of action
   - protein tyrosine kinase: can phosphorylate itself, other proteins at tyrosine residues
      - autophosphorylation: increases activity of tyrosine kinase
      - phosphorylaton: occurs on insulin receptor substrates 1 and 2 (IRS-1, IRS-2)
   - phosphorylated IRS-1, IRS-2: can serve as docking sites for multiple effector molecules
      - adaptor proteins
      - protein kinases
      - phospholipid kinases
      - phosphatases
   - this interaction helps regulate cellular function
- downregulation of the insulin-receptor complex: internalization by endocytosis, with two fates
   - degradation: fusion with lysosomes (linked to receptor downregulation in hyperinsulinemic states)
   - recycling:      return to the plasma membrane (linked to insulin clearance)
- mechanisms of signal transduction
   - tyrosine kinase: phosphorylates various proteins that amplify the insulin signal
   - phosphatidylinositol 3-kinase: insulin-induced glucose uptake with some glucose transporters
   - cAMP: decreased in liver, adipose by activation of cAMP phosphodiesterase, inhibition of adenylate cyclase (G i)
insulin disease states
- hypoinsulinism (diabetes mellitus): disorder of CHO, protein, or lipid metabolism
   - classic symptoms: hyperglycemia, glycosuria (though these symptoms are found in numerous other diseases)
   - central cause: abnormality in the synthesis, secretion, and/or effect of insulin
      - insulin-receptor interaction is an important component of insulin’s effects
      - diabetes mellitus can be caused by defects at this stage, through receptor deficiencies or autoimmunity
- hyperinsulinism: excessive insulin production or overdose
   - consequence: severe reduction in blood glucose, due to uptake by muscle and adipose
   - symptoms: CNS disturbances, convulsions, coma, and death
- note: hypoinsulinism is tolerated much better than hyperinsulinism




55. Pancreatic Hormones – Glucagon
Study Guide
glucagon
- glucagon chemistry
   - structure: single chain polypeptide (29 AA)
   - synthesis: produced in the α cells of the endocrine pancreas
- regulation
   - process: similar to insulin; contained in granules in α cells, released by exocytosis
   - factors affecting release
      - low blood glucose: primary regulation; low blood glucose leads to low intracellular concentration in α cells
      - increased blood AA: allows amino acids to be used as gluconeogenic substrates
                                                                       Biochemistry: NOTES & OBJECTIVES (page 127 of 165)



      - insulin: decreases glucagon secretion (decreased intracellular cAMP, downregulation of glucose entry)
      - catecholamines: cause increased glucagon release (β-adrenergic receptors)
- biological effects
   - stimulates glycogenolysis: increases glycogen phosphorylase, decreases glycogen synthase; no effect in muscle
   - inhibits liver glycolysis: downregulates fructose 2,6-bisphosphate production, inactivates pyruvate kinase
   - promotes adipose lipolysis: enhances activity of hormone-sensitive lipase via cAMP-PKA
   - stimulates gluconeogensis: increases utilization of gluconeogenic amino acids, synthesis of enzymes
   - promotes AA catabolism: increases synthesis of key AA enzymes, urea cycle enzymes
- signal transduction
   - Gs system: activation of protein kinase A (PKA)
      - glucagon binds Gs protein, activates adenylate cyclase, increases cAMP
      - cAMP causes activation of PKA, which then mediates the hormonal effects via phosphorylations
   - Gq system: activation of protein kinase C (PKC), release of intracellular Ca 2+
      - glucagon binds Gq protein, activates PLC, which catalyzes PIP 2  IP3 + DAG
         - IP3: causes release of Ca2+ from intracellular stores
         - DAG: activates protein kinase C
      - PKC, Ca2+ mediate the hormonal effects
   - glucagon second messenger summary
      - cAMP: inhibits glycogen synthesis (PKA inhibition of glycogen synthase)
                 stimulates glycogen degradation (PKA activation of phosphorylase kinase)
      - DAG: inhibits glycogen synthesis (PKC inhibition of glycogen synthase)
      - Ca2+: stimulates glycogen degradation (Ca2+-calmodulin activation of phosphorylase kinase)
- insulin-glucagon interactions
   - general response
      - catabolic:             low I/G (favoring mobilization of nutrient stores, enhanced protein and FA catabolism)
      - anabolic:              high I/G (favoring nutrient storage and anabolism)
   - relationships: glucose tolerance tests with glucose alone, gelatin alone relative to a normal meal
      - gelatin and glucose meal (representative of a typical meal)
         - glucose:            spikes, returns to normal
         - insulin:            spikes (before glucose), returns to normal
         - glucagon:           increases, levels off
         - I/G:                increases, returns to normal
      - glucose alone
         - glucose:            same
         - insulin:            same
         - glucagon:           unlike gelatin/glucose meal, does not change much
         - I/G:                same
      - gelatin alone
         - glucose:            unlike gelatin/glucose meal, does not change much
         - insulin:            increases only slightly
         - glucagon:           increases above that of the gelatin/glucose meal
         - I/G ratio:          unlike gelatin/glucose meal, does not change much


Notes: Lecture and Reading
glucagon chemistry
- site of synthesis: α cells of the islets of Langerhans in the endocrine pancreas
- chemistry: single chain polypeptide (29 AA)
glucagon storage and release
- storage: contained in granules in α cells, released by exocytosis
- factors affecting release
   - low blood glucose: primary regulation; low blood glucose leads to low intracellular concentration in α cells
   - increased blood AA: allows amino acids to be used as gluconeogenic substrates
   - insulin: decreases glucagon secretion (decreased intracellular cAMP, downregulation of glucose entry)
   - catecholamines: cause increased glucagon release
metabolic effects of glucagon
                                                                  Biochemistry: NOTES & OBJECTIVES (page 128 of 165)



- stimulates glycogenolysis (rapid)
   - increases glycogen phosphorylase activity
   - decreases glycogen synthase (via cAMP-dependent PKA pathway)
   - note: glucagon does NOT affect muscle glycogen phosphorylase!!!
- inhibits liver glycolysis (rapid)
   - downregulates fructose 2,6-bisphosphate production, causing decreased PFK-1
   - promotes phosphorylation, inhibition of pyruvate kinase via PKA
- promotes lipolysis in adipose tissues (rapid)
   - enhances activity of hormone-sensitive lipase via cAMP-PKA
- stimulates gluconeogensis (slow)
   - increases utilization of gluconeogenic amino acids
   - increases synthesis of key gluconeogenic enzymes (e.g. PEPCK) via cAMP-dependent transcription (CREB)
- promotes amino acid catabolism (slow)
   - increases synthesis of key enzymes in AA metabolism (e.g. serine dehydratase, transaminases)
   - increases synthesis of urea cycle enzymes
mechanism of action
- Gs system: activation of protein kinase A (PKA)
   - glucagon binds Gs protein, causing activation of adenylate cyclase
   - adenylate cyclase catalyzes formation of cAMP
   - cAMP causes activation of PKA, which then mediates the hormonal effects via phosphorylation
- Gq system: activation of protein kinase C (PKC), release of intracellular Ca 2+
   - glucagon binds Gq protein, causing activation of phospholipase C (PLC)
   - PLC catalyzes conversion of PIP2 to IP3 and DAG
      - IP3: causes release of Ca2+ from intracellular stores
      - DAG: activates protein kinase C
   - PKC, Ca2+ mediate the hormonal effects
- glucagon receptor interactions
   - cAMP: inhibits glycogen synthesis (PKA inhibition of glycogen synthase)
                 stimulates glycogen degradation (PKA activation of phosphorylase kinase)
   - DAG: inhibits glycogen synthesis (PKC inhibition of glycogen synthase)
   - Ca2+:       stimulates glycogen degradation (Ca2+-calmodulin activation of phosphorylase kinase)
insulin-glucagon interactions
- insulin/glucagon: primary ratio affecting nutrient balance under normal conditions
- typical I/G ratios
   - glucose meal:             I/G ~ 40.0
   - glucose infusion (IV): I/G ~ 16.0
   - overnight fast:           I/G ~ 4.0
   - starvation:               I/G ~ 0.4
- general responses
   - catabolic:      low I/G (favoring mobilization of nutrient stores, enhanced protein and FA catabolism)
   - anabolic:       high I/G (favoring nutrient storage and anabolism)
- relationships: glucose tolerance tests with glucose alone, gelatin alone relative to a normal meal
   - gelatin and glucose meal (representative of a typical meal)
      - glucose:     spikes, returns to normal
      - insulin:     spikes (before glucose), returns to normal
      - glucagon: increases, levels off
      - I/G:         increases, returns to normal
   - glucose alone
      - glucose:     same
      - insulin:     same
      - glucagon: unlike gelatin/glucose meal, does not change much
      - I/G:         same
   - gelatin alone
      - glucose:     unlike gelatin/glucose meal, does not change much
      - insulin:     increases only slightly
      - glucagon: increases above that of the gelatin/glucose meal
                                                                       Biochemistry: NOTES & OBJECTIVES (page 129 of 165)



    - I/G ratio:   unlike gelatin/glucose meal, does not change much




56. Adrenal Hormones – Epinephrine and Cortisol
Study Guide
adrenaline
- catecholamine chemistry
   - norepinephrine
      - structure:             C6H4(OH)2-CH(OH)-CH2-NH3+                     (non-methylated epinephrine)
      - function:              principally a neurotransmitter of the sympathetic nervous system
   - epinephrine
      - structure:             C6H4(OH)2-CH(OH)-CH2-NH-CH3                   (OH: positions 3, 4)
      - function:              principal catecholamine of energy metabolism
- catecholamine biosynthesis
   - reactions
      - tyrosine hydroxylase:                               tyrosine  dihydrophenylalanine (L-DOPA)
      - DOPA decarboxylase:                                 L-DOPA  dopamine
      - dopamine β-hydroxylase:                             dopamine  norepinephrine
      - phenylethanolamine N-methyltransferase:             norepinephrine  epinephrine
   - key regulatory point
      - enzyme: phenylethanolamine N-methyltransferase (PMNT)
      - regulation: glucocorticoids (cortisol) induce synthesis of PMNT
- adrenaline storage and release
   - localization: cytoplasmic granules of the chromaffin cells
   - signal:          controlled by nerve stimulation (stress, hypotension, anoxia), profound hypoglycemia
   - secretion:       acetylcholine: mobilizes Ca2+, promoting epinephrine release from granules
- adrenaline receptors
   - α1 receptor: Gq-mediated PLC activation, used in smooth muscle contraction
   - α2 receptor: Gi-mediated decrease in cAMP, suppressing insulin release from pancreatic β cells
   - β receptors: Gs-mediated increase in cAMP, activation of PKA; similar glucagon effects, but works on muscle
- adrenaline biological effects
   - non-metabolic
      - α-adrenergic stimulators: vasoconstriction
      - β-adrenergic stimulators: increased ventilation, blood flow, heart rate, and peripheral vessel dilation
   - metabolic
      - stimulates glycogenolysis:
         - muscle: increased even at low adrenergic levels (β: ↑ cAMP, active PKA)
         - liver: increased at high levels of stress (β: ↑ cAMP, active PKA)
         - non-cAMP-dependent glycogen degradation (α1: Gq, ↑ PLC, ↑ Ca2+ and DAG giving active PKC)
      - stimulates lipolysis
         - activates hormone-sensitive lipase (β: Gs, ↑ cAMP, active PKA)
         - glucocorticoids: permissive effect on hormone-sensitive lipase
      - enhances gluconeogenesis: induces PEP carboxykinase
      - inhibits insulin release: suppresses cAMP production in pancreatic β cells (α2: Gi, ↓ cAMP, inactive PKA)
- adrenaline regulation
   - T3, T4: increase number, effectiveness (decrease Gi synthesis) of membrane receptors
      - hyperthyroidism: β-adrenergic hypersensitivity (Gs effects relatively stronger)
      - hypothyroidism: β-adrenergic hyposensitivity (Gi effects relatively stronger)
   - catabolism: monoamine oxidase (MAO), catechol-O-methyltransferase (COMT)
cortisol
- cortisol biosynthesis: synthesized in the adrenal cortex
- cortisol regulation
                                                                      Biochemistry: NOTES & OBJECTIVES (page 130 of 165)



   - transcriptional: primary regulation, under the control of the hypothalamus and pituitary glands
   - release: enhanced during times of stress (e.g. starvation)
   - timing: regulated on a circadian rhythm, peaking in the morning upon waking up
- cortisol biochemical effects
   - by effect
      - stimulates gluconeogenesis
         - inhibits general protein synthesis, stimulates protein degradation
         - inhibits uptake of amino acids by muscle, other peripheral tissues
         - induces liver AA-catabolizing enzymes of gluconeogenic amino acids
         - induces synthesis of several liver gluconeogenic enzymes
      - permissive effect on lipolysis, glycogenolysis
         - mechanism: not well understood, though at high concentrations, can lead to glycogen deposition in liver
         - glycogen deposition mechanism: large increase in glucose 6-phosphate levels via its promotion of GNG
      - inhibits insulin-induced glucose uptake in muscle, adipose tissues
   - by nutrient
      - carbohydrate:          ↑ gluconeogenesis
                               ↓ insulin uptake in peripheral tissues
      - protein:               ↑ degradation, ↓ synthesis in muscle
                               ↑ concentration of circulating AA
                               ↑ synthesis of gluconeogenic enzymes in liver
                               ↑ ureogenesis
      - fat:                   ↑ peripheral lipolysis
                               ↑ circulating lipids
                               centralization of fat distribution (bringing it into the liver)


Notes: Lecture and Reading
introduction: adrenal hormones
- adrenal gland composition
   - cortex
   - medulla
- hormones produced in the areas of the adrenal structures differ in effects, but cooperate to influence metabolism
- adrenal defects: most common endocrine disorder in humans
adrenal medulla: norepinephrine, epinephrine
- adrenaline (catecholamine) chemistry
   - norepinephrine
      - structure:          C6H4(OH)2-CH(OH)-CH2-NH3+                     (non-methylated epinephrine)
      - prevalence:         less common than epinephrine (16% of catecholamine total)
      - function:           principally a neurotransmitter of the sympathetic nervous system
      - half life:          20-40 seconds
      - synthesis:          produced in the adrenal medulla, adrenergic nervous tissue, and the brain
   - epinephrine
      - structure:          C6H4(OH)2-CH(OH)-CH2-NH-CH3                   (OH: positions 3, 4)
      - prevalence:         most common catecholamine
      - function:           principal catecholamine of energy metabolism
      - half life:          20-40 seconds
      - synthesis:          produced in the adrenal medulla via phenylethanolamine N-methyl transferase (PNMT)
- catecholamine biosynthesis
   - reactions (NOT balanced equations)
      - tyrosine hydroxylase:                            tyrosine  dihydrophenylalanine (L-DOPA)
      - DOPA decarboxylase:                              L-DOPA  dopamine
      - dopamine β-hydroxylase:                          dopamine  norepinephrine
      - phenylethanolamine N-methyltransferase:          norepinephrine  epinephrine
   - key regulatory point
      - enzyme: phenylethanolamine N-methyltransferase (PMNT)
      - regulation: glucocorticoids (cortisol) induce synthesis of PMNT
                                                                        Biochemistry: NOTES & OBJECTIVES (page 131 of 165)



- adrenaline storage and release
   - localization: cytoplasmic granules of the chromaffin cells
   - signal:           controlled by nerve stimulation (stress, hypotension, anoxia) and profound hypoglycemia
                       - hypotension: low blood pressure
                       - anoxia: low O2 content in blood
   - exocytosis: acetylcholine: mobilizes Ca2+, promoting epinephrine release from granules
                       acetylcholine regulation: regulated by a stress-linked signal at the hypothalamus
- biochemical effects and mechanism of action
   - α, β effects
      - biological effects: classified as α or β effects
      - substances: classified as α- or β-adrenergic, with target tissues having these plasma membrane receptors
      - the classification is not absolute
         - norepinephrine: pure α stimulator
         - epinephrine:         primarily β stimulator, but also has α effects at high concentrations
      - there are no natural pure β stimulators
   - receptor classes for epinephrine
      - α1 receptor
         - G protein:           Gq
         - mechanism:           activation of PLC, producing IP3 (↑ Ca2+) and DAG (active PKC)
         - function:            smooth muscle contraction
      - α2 receptor
         - G protein:           Gi
         - mechanism:           inhibition of adenylate cyclase, reduction of cAMP, inactive PKA
         - function:            in β cells, suppresses insulin release to reserve glucose for CNS, skeletal muscle
      - β receptors (β1, β2, β3)
         - G protein:           Gs
         - mechanism:           activation of adenylate cyclase, increase in cAMP, active PKA
         - function:            similar to glucagon in liver, adipose tissue; also works on muscle
   - specific biological effects
      - non-metabolic effects
         - α-adrenergic stimulators: vasoconstriction
         - β-adrenergic stimulators: increased ventilation, blood flow, heart rate, and peripheral vessel dilation
      - metabolic effects
         - stimulates glycogenolysis
            - muscle: increased even at low adrenergic levels (β: ↑ cAMP, active PKA)
            - liver: increased at high levels of stress (β: ↑ cAMP, active PKA)
            - non-cAMP-dependent glycogen degradation (α1: Gq, ↑ PLC, ↑ Ca2+ and DAG giving active PKC)
         - stimulates lipolysis
            - activates hormone-sensitive lipase (β: Gs, ↑ cAMP, active PKA)
            - glucocorticoids: permissive effect on hormone-ensitive lipase
         - enhances gluconeogenesis
            - induces PEP carboxykinase
         - inhibits insulin release
            - suppresses cAMP production in pancreatic β cells (α2: Gi, ↓ cAMP, inactive PKA)
      - thyroid hormone (T3, T4) regulation of β-adrenergic effects
         - mechanism:           increase number of receptors in membrane
                                modify receptor effectiveness (e.g. decrease synthesis of G i, giving more cAMP)
         - defects:             hyperthyroidism: β-adrenergic hypersensitivity (Gs effects relatively stronger)
                                hypothyroidism: β-adrenergic hyposensitivity (Gi effects relatively stronger)
- catecholamine catabolism
   - overview: half lives
      - catecholamine half life: 20-40 seconds
      - thyroxine half life: ~7 days
      - extending catecholamine half lives can lead to heart attack
   - catecholamine breakdown: enzymes
      - monoamine oxidase (MAO)
                                                                        Biochemistry: NOTES & OBJECTIVES (page 132 of 165)



         - mechanism:         oxidative deamination
         - function:          inactivation of catecholamines in nerve endings
         - localization:      CNS, kidney, liver
      - catechol-O-methyltransferase (COMT)
         - mechanism:         O-methylation
         - function:          inactivation of circulating catecholamines
         - localization:      plasma, liver, kidney, red blood cells
adrenal cortex: cortisol
- cortisol biosynthesis: synthesized in the adrenal cortex
- cortisol regulation
   - transcriptional: primary regulation, under the control of the hypothalamus and pituitary glands
   - release: enhanced during times of stress (e.g. starvation)
   - timing: regulated on a circadian rhythm, peaking in the morning upon waking up
- biochemical effects and mechanism of action
   - stimulates gluconeogenesis
      - inhibits general protein synthesis, stimulates protein degradation
      - inhibits uptake of amino acids by muscle, other peripheral tissues
      - induces liver AA-catabolizing enzymes of gluconeogenic amino acids
      - induces synthesis of several liver gluconeogenic enzymes
   - permissive effect on lipolysis, glycogenolysis
      - mechanism: not well understood, though at high concentrations, can lead to glycogen deposition in liver
      - glycogen deposition mechanism: large increase in glucose 6-phosphate levels via its promotion of GNG
   - inhibits insulin-induced glucose uptake in muscle, adipose tissues
- summary: major metabolic actions of cortisol on CHO, protein, and fat
   - carbohydrate:            ↑ gluconeogenesis
                              ↓ insulin uptake in peripheral tissues
   - protein:                 ↑ degradation, ↓ synthesis in muscle
                              ↑ concentration of circulating AA
                              ↑ synthesis of gluconeogenic enzymes in liver
                              ↑ ureogenesis
   - fat:                     ↑ peripheral lipolysis
                              ↑ circulating lipids
                              centralization of fat distribution (bringing it into the liver)




57. Hormonal Control of Energy Metabolism
Study Guide
- tissue roles in energy metabolism
   - skeletal muscle: movement
      - energy use
         - reduced energy anabolism: lacks gluconeogenesis, pentose phosphate pathway, glycerol kinase
         - increased energy catabolism: able to utilize hexoses, fatty acids, pyruvate, ketone bodies
      - metabolic activity
         - anabolic conditions: highly active glycolysis, TCA, and O2 consumption
         - catabolic conditions: highly active protein catabolism (majority of SS utilized by gluconeogenesis)
   - liver: metabolic hub
      - diversity: can take in glucose and AA, export glucose and FA (as TG)
      - adaptability: able to alter enzyme content quickly, allowing rapid adaptibility to dietary alterations
      - metabolic activity
         - glycogen metabolism: serves as a readily accessible store of glucose
         - gluconeogenesis: major site during short term fast
         - fat synthesis: contains glycerol kinase (allowing it to synthesize fat without concurrent glycolysis)
                                                                      Biochemistry: NOTES & OBJECTIVES (page 133 of 165)



   - adipose tissue: energy store
      - function: metabolic resiliency, providing FA on demand to meet energy needs
      - metabolic activity
         - does very little work, and thus requires very little metabolism
         - low TCA, minimal glycogen, no gluconeogenesis, no glycerol kinase, active pentose phosphate pathway
   - nervous tissue: philosophy
      - metabolic substrates
         - anabolic conditions: glucose, mannose (primary); glycerol, lactate, butyrate, some amino acids (secondary)
         - catabolic conditions: ketone bodies (brain adapts through change in enzyme profile
      - metabolic activity: high rate, with little glycogen, so quite dependent on blood O 2 and glucose
   - red blood cells: O2 transport
      - function: carry O2 to various tissues, deal with problems associated with O2 transport
      - metabolic activity: no TCA cycle, active pentose phosphate
- catabolic state
   - hormonal profile: low I/G, elevated cortisol/adrenaline (depending on stress)
    - carbohydrate catabolism: active glycogenolysis, glycolysis, gluconeogenesis
      - glycogen degradation: active phosphorylase kinase, inactive glycogen synthase
      - glycolysis: decreased F26BP ( decreased PFK-1), decreased pyruvate kinase
      - gluconeogenesis: decreased PFK, pyruvate oxidation, increased synthesis of GNG enzymes
   - fatty acid oxidation: decreased; low acetyl-CoA carboxylase, low malonyl-CoA
   - ketone body formation: increased; high FA-CoA forms into KB by mass action
   - AA catabolism: low insulin, low resynthesis, increased hepatic uptake and breakdown
- anabolic state
   - hormonal profile: high I/G, low cortisol/adrenaline (depending on stress)
   - carbohydrate catabolism: increased glycogen; decreased phosphorylase, increased synthase (low cAMP)
   - lipogenesis: highly active TG synthesis
      - fatty acid synthesis: increased; active carboxylase, increased substrates, high malonyl-CoA
      - triglyceride export from liver: insulin (or glucocorticoid) activation of VLDL synthesis
   - protein synthesis: increased; increased AA entry into muscle, protein synthesis; insulin permissive to GH
- diabetes mellitus
   - metabolic disturbances
      - liver
         - glycogen breakdown:          increased (despite high blood glucose)
         - gluconeogenesis:             increased
         - β-oxidation:                 increased, leading to elevated acetyl-CoA and ketosis
      - muscle
         - glycogen synthesis:          decreased
         - glucose oxidation:           decreased
         - protein synthesis:           decreased
         - protein breakdown:           increased
      - adipose
         - TG synthesis:                decreased
         - TG breakdown:                increased
      - water, electrolyte balance
         - hyperglycemia:                glycosuria, ketoacidosis, ketonuria
   - type I: insulin-dependent (juvenile) diabetes
      - onset:       abrupt
      - nutrition: malnourished
      - insulin:     deficient; required for all patients
      - symptoms: polydypsia, polyphagia, polyuria
      - ketosis:     frequent; helps distinguish from type II
   - type II: insulin-resistant (adult) diabetes
      - onset:       gradual
      - nutrition: obese
      - insulin:     present and excessive; not as frequently needed in treatment
      - symptoms: frequently none or mild
                                                                        Biochemistry: NOTES & OBJECTIVES (page 134 of 165)



    - ketosis:    rare; helps distinguish from type I


Notes: Lecture and Reading
metabolic logic
- metabolic signals
   - blood glucose
   - blood amino acids
   - stress
- hormonal profile
   - anabolic: insulin
   - catabolic: glucagon, adrenaline, cortisol
   - developmental: growth hormone, thyroid-stimulating hormone, sex steroids
- key cell, tissue types
   - liver
   - adipose tissue
   - skeletal muscle
   - CNS
   - red blood cells
review of metabolic profiles and functions of key cell types
- skeletal muscle: movement
   - energy anabolism: reduced abilities
      - lacks glucose 6-phosphatase, other key gluconeogenic enzymes
      - lacks the pentose phosphate pathway
      - lacks glycerol kinase (useful in triglyceride synthesis)
   - energy catabolism: numerous sources
      - hexoses
      - fatty acids
      - pyruvate
      - ketone bodies
   - metabolic activity
      - O2 consumption
         - resting: 30% of O2 consumption
         - exercising: up to 80% of O2 consumption
      - glycolysis: highly active
      - TCA cycle: highly active
      - glycogenolysis: activity dependent on need; cannot export glucose as does the liver
      - protein catabolism
         - during fasting, stress, skeletal muscle highly active in protein breakdown
         - accounts for the majority of amino acids utilized by hepatic/renal gluconeogenesis
   - differences in cardiac muscle
      - function: must work continuously, and is more dependent on aerobic metabolism
      - ultrastructure: higher levels of mitochondria, myoglobin
- liver: metabolic hub
   - adaptability
      - adapts rapidly to change in diet
      - able to alter enzyme content quickly
   - metabolic activity
      - glycogen metabolism: serves as a readily accessible store of glucose
      - gluconeogenesis: major site during short term fast
      - fat synthesis: contains glycerol kinase (allowing it to synthesize fat without concurrent glycolysis)
   - metabolite import and export: liver as a central hub of metabolism
      - uptake: glucose without hormonal control, amino acids with hormonal facilitation
      - export: glucose, fatty acids (as TG)
- adipose tissue: energy store
   - function
                                                                      Biochemistry: NOTES & OBJECTIVES (page 135 of 165)



      - provides metabolic resiliency through efficient energy storage as triglyceride
      - can provide fatty acids on demand
   - metabolic activity: does very little work, and thus requires very little metabolism
      - TCA: low
      - glycogen storage: minimal
      - gluconeogenesis: none
      - fat synthesis: no glycerol kinase (requires glycolysis to provide glycerol phosphate)
      - pentose phosphate: highly active, used in FA biosynthesis
- nervous tissue: makes you think and stuff
   - metabolic substrates: depends largely on blood-brain barrier
      - primary fuel: glucose, mannose
      - secondary fuels: glycerol, lactate, butyrate, some amino acids
      - fasting: ketone bodies (brain adapts through change in enzyme profile
   - metabolic activity
      - overall metabolism: occurs at a high rate, so glucose and O2 exhausted in minutes
      - glycogen storage: minimal, thus giving high dependence on blood glucose levels
- red blood cells: O2 transport
   - function: carry O2 to various tissues, deal with problems associated with O2 transport
   - metabolic activity
      - TCA cycle: absent (depend entirely on anaerobic glycolysis)
      - pentose phosphate: active (produces NADPH which is used to deal with oxidative problems)
metabolic overview
- metabolic status: catabolic vs. anabolic
   - anabolic
      - function: biosynthesis of cellular macromolecules
      - examples: protein, glycogen, FA, TG
   - catabolic
      - function: breakdown of complex molecules to provide ATP, reducing equivalents
      - examples: protein degradation, glycogenolysis, lipolysis, FA oxidation
      - gluconeogenesis: biosynthetic process that is part of catabolic state, due to CNS glucose need
- hormonal balance between catabolic, anabolic states
   - insulin/glucagon (I/G): important in determining anabolic vs. catabolic state (regulated by blood glucose)
      - insulin: major anabolic hormone, promoting CHO utilization, nutrient storage
      - glucagon: major catabolic hormone, promoting utilization of stored nutrients and gluconeogenesis
   - other important hormones
      - stress: cortisol, adrenaline
      - development: growth hormone
catabolic state
- hormonal profile
   - insulin:                  depressed
   - glucagon:                 elevated
   - adrenaline, cortisol: possibly elevated (depending on stress)
   - growth hormone:           possibly elevated

- carbohydrate catabolism: glycogenolysis, glycolysis
   - glycogen degradation
      - regulation:       low I/G; high adrenaline
      - mechanism:        Gs activation leading to increased cAMP, PKA
                          Gq activation leading to increased Ca2+, PKC
      - effects:          activation of phosphorylase kinase (and glycogen phosphorylase)
                          inhibition of glycogen synthase
   - glycolysis
      - regulation:       low I/G; high adrenaline
      - mechanism:        Gs activation leading to increased cAMP, PKA
      - effects:          decrease in F26BP (and thus decreased phosphofructokinase-1)
                          decrease in pyruvate kinase
                                                                      Biochemistry: NOTES & OBJECTIVES (page 136 of 165)



  - gluconeogneesis
     - regulation:          low I/G; high cortisol
     - mechanism:           Gs activation leading to increased cAMP, PKA
                            transcriptional inactivation of gluconeogenic enzymes
    - effects:              decreased phosphofructokinase (lessens glucose use)
                            increased pyruvate oxidation (increases lactate, alanine, pyruvate as GNG substrates)
                            increased synthesis of gluconeogenic enzymes
                            - glucose 6-phosphatase
                            - fructose 1,6 bisphosphatase
                            - phosphoenolpyruvate carboxykinase
   - fatty acid oxidation
      - regulation:        low I/G; uptake controlled by blood levels
      - mechanism:         Gs activation leading to increased cAMP, PKA, decreased acetyl-CoA carboxylase
      - effects:           low malonyl-CoA (leading to increased FA mitochondrial uptake, oxidation)
   - ketone body formation
      - regulation:        NADH/NAD, high blood FA (leading to high FA-CoA in the cell)
      - mechanism:         elevated FA-CoA: inhibits TCA cycle, leads to increased acetyl-CoA
      - effects:           acetyl-CoA diverted to ketone body synthesis (NADH/NAD, mass action)
      - utilization:       initially used by muscle, heart, kidney; later used by brain to spare glucose
- protein/amino acid catabolism
   - regulation:           low insulin, high glucagon (liver), high cortisol and adrenaline (muscle, liver)
   - mechanism:            glucagon, cortisol: elevate levels of catabolic proteins, hepatic AA uptake
                           low insulin: allows decreased protein biosynthesis
   - effects:              net protein breakdown into AA
                           AA transport to liver for gluconeogenesis, detoxification
anabolic state
- hormonal profile
   - insulin:              elevated
   - glucagon:             depressed
   - adrenaline, cortisol: possibly depressed (depending on stress)
   - growth hormone:       elevated

- glycogen synthesis
   - regulation:            high I/G
   - mechanism:             Gs inactivation leading to low cAMP, low PKA
                            insulin-dependent activation of cAMP phosphatase (lower cAMP, low PKA)
  - effects:                decreased glycogen phosphorylase
                            increased glycogen synthase
- lipogenesis
   - fatty acid synthesis
      - regulation:          high I/G
      - mechanism:           activation of acetyl-CoA carboxylase (Gs and ↓ cAMP; also insulin-dependent kinase)
                             increased FA synthesis substrates
                             - I/G increases in glycolysis, decreases in gluconeogenesis
                             - insulin-dependent activation of PDH, high acetyl-CoA, giving high citrate
                             - insulin induction of lipogenic enzymes, pentose phosphate enzymes
      - effects:             increased cytoplasmic acetyl-CoA, leading to malonyl-CoA and FA biosynthesis
   - triglyceride synthesis and export from liver
      - regulation:          high I/G
      - mechanism:           fed: insulin activation of VLDL synthesis; FFA  TG in adipose
                             stressed: glucocorticoid activation of VLDL synthesis; FFA  CO2 in muscle
      - effects:             decreased TG breakdown
                             increased TG synthesis by mass action
                             increased synthesis and secretion of VLDL
- protein synthesis
   - regulation:             increased insulin, increased glucagon
                                                                         Biochemistry: NOTES & OBJECTIVES (page 137 of 165)



  - mechanism:                 elevated blood AA stimulation of insulin, glucagon (even in absence of CHO)
  - effects:                   increased AA entry into muscle
                               increased protein synthesis in muscle, liver
   - note: insulin is also permissive to growth hormone, which acts similarly to insulin
disease states: diabetes mellitus
- overview
   - diabetes mellitus: deficiency in insulin synthesis, secretion, or effects leading to exaggerated catabolic state
   - insulin activity: artificially low, leading to loss of glucose uptake in peripheral tissues
   - glucagon: dominates, leading to very low I/G and the catabolic condition
- metabolic disturbances
   - liver
      - glycogen breakdown:              increased (despite high blood glucose)
      - gluconeogenesis:                 increased
      - β-oxidation:                     increased, leading to elevated acetyl-CoA and ketosis
   - muscle
      - glycogen synthesis:              decreased
      - glucose oxidation:               decreased
      - protein synthesis:               decreased
      - protein breakdown:               increased
   - adipose
      - TG synthesis:                    decreased
      - TG breakdown:                    increased
   - water, electrolyte balance
      - hyperglycemia:                    glycosuria, ketoacidosis, ketonuria

TABLE: Diabetes Mellitus, Type I and Type II
 parameter            type I                                                   type II
 other names          insulin-dependent                                        insulin-resistant
                      juvenile onset                                           adult onset
 age of onset         generally under 35                                       generally over 35
 type of onset        abrupt: days to weeks, often following an                gradual: weeks to months
                      illness
 nutritional status   generally undernourished                                 generally obese
 (at onset)
 endogenous insulin   negligible to absent                                     present, even excessive, but ineffective
                                                                               due to obesity
 symptoms                 polydypsia                                           frequently none or mild
                          polyphagia
                          polyuria
 ketosis                  frequent: major diagnostic tool for type I           infrequent: insulin usually sufficient to
                                                                               prevent lipolysis
 insulin treatment        needed for all patients                              necessary only in about 20-30% of
                                                                               patients; diet alone can usually control
                                                                               blood glucose
  - hemoglobin A1c
     - glycosylated Hb form
     - higher than normal levels can be indicative of recent transient high blood glucose (e.g. binge eating)


SUPPLEMENTARY TABLE: Biochemican Indications and Their Causes in Diabetes Mellitus
 indication    cause
 hyperglycemia decreased glucose uptake by peripheral tissues
               increased hepatic glycogen mobilization
               increased hepatic gluconeogenesis
 glycosuria    glucose load exceeds capacity for reabsorption in renal tubule
                                                                         Biochemistry: NOTES & OBJECTIVES (page 138 of 165)



 ketoacidosis              increased β-oxidation of adipose tissue fatty acids in liver, leading to elevated hepatic
                           acetyl-CoA concentrations and ketone body synthesis
 ketonuria                 ketone load exceeds capacity for reabsorption in renal tubule
 hyperlactatemia           mobilization, metabolism of muscle glycogen to lactate, and lactate released as precursor
                           of gluconeogenesis (Cori cycle)
 hyperlipidemia            free fatty acids derived from increased lipolysis in adipose tissue
 hypertryglyceridemia      increased synthesis of triglyceride in liver, and increased VLDL synthesis
 hypevolemia /             excessive loss of body water as urine due to glucose acting as an osmotic diuretic
 hyperosmolarity
 hyponatremia              loss of body sodium, as a result of glucose-induced osmotic diuresis


SUPPLEMENTARY TABLE: Clinical Symptoms and Their Causes in Diabetes Mellitus
 symptom          cause
 polyuria         retention of glucose in renal tubule as glucose load exceeds absorptive capacity; glucose
                  acts as an osmotic diuretic, producing large volumes of urine
 polydypsia       CNS-driven response to dehydration; may be mediated by angiotensin produced in
                  resopnse to hypovolemia
 polyphagia       hunger stimulated by non-utilization of dietary glucose
 weight loss      increased catabolism of all metabolic fuel stores: muscle glycogen, protein, adipose TG
 tiredness        muscular weakness due to:
                  - proteolysis and mobilization of muscle protein
                  - reduced availability of metabolic substrate
 blurred vision   dehydration of the lens, aqueous and vitreous humor; cataracts from sorbitol accumulation
 vomiting         CNS-driven response to ketones stimulating area postrema in floor of the fourth ventricle
 hyperventilation respiratory compensation to metabolic acidosis (elevated plasma lactate, keto acids)
 itching          hyperosmotic blood leads to water loss from the skin




58. Hypothalamus: Hypothalamic Releasing Factors and Posterior
       Pituitary Hormones
Study Guide
hypothalamic releasing factors
- synthesis, release, and effects
   - synthesis:     hypothalamus
   - transport:     hypothalamic-hypophysial portal system of capillaries
   - target:        anterior pituitary hormones
   - function:      act at the level of the anterior pituitary to regulate hormone synthesis, secretion
- hypothalamic hormones and their targets
   - corticotropin-releasing factor (CRF):                  ACTH
   - thyrotropin-releasing hormone (TRH):                   TSH, prolactin
   - gonadotropin releasing hormone (GnRH):                 LH, FSH
   - somatostatin:                                          GH, TSH, glucagon, insulin, gastrin, secretin, others
   - growth hormone releasing factor (GH-RF):               GH
   - prolactin release-inhibiting factor (PIF):             prolactin
   - prolactin releasing factor (PRF):                      prolactin
   - melanocyte-stimulating hormone IF:                     MSH
   - melanocyte-stimulating hormone RF:                     MSH
- general chemistry
   - chemistry: most are peptides; PIF is dopamine (catecholamine)
                                                                        Biochemistry: NOTES & OBJECTIVES (page 139 of 165)



   - effects: primarily regulate tropic hormone secretion; secondarily regulate synthesis
   - receptors: specific receptors for factors on the surface of specific cell types
   - mechanism: act via changes in intracellular Ca2+, PI metabolism, and/or cAMP
      - CRF:         enhances cAMP production in cells
      - GnRH:        elevate intracellular Ca2+ via production of IP3 (release from ER) and DAG ( PKC)
      - TRH:         elevate intracellular Ca2+ via production of IP3 (release from ER) and DAG ( PKC)
- regulatory mechanism
   - general
      - releasing factors: secreted from hypothalamus as a result of extrahypothalamic or neural stimuli
      - tropic hormones: release/synthesis in anterior pituitary as a result of releasing factors
      - end organ hormones: final hormones produced in response to tropic hormones; generally feed back
   - example: feedback control by the gonads
      - extrahypothalamic stimuli (drugs, stress): stimulate hypothalamus
      - hypothalamus: releases GnRH (releasing factor) into portal system
      - anterior pituitary: releases FSH, LH (tropic hormones) into bloodstream
      - gonads (target organ): produce gonadal steroids (end organ hormones), which cause the metabolic effect
      - gonadal steroids: negatively feed back on anterior pituitary, hypothalamus
oxytocin
- function:          major hormone of ejection (babies, milk, vomit, sexual fluids)
- synthesis:         single gene product (9 AA oligopeptide)
- processing:        cleaved into neurophysin I and hormone, transported to axons of the posterior pituitary
- secretion:         released as a hormone-neurophysin I complex
- effects:           females: stimulates contraction of the uterus and the mammary alveoli
                     - permissive effects: requires estrogen priming, lactating conditions
                     males: inhibits androgen biosynthesis in testis (mechanisms, logic are unclear)
                     others: may also have effects on satiety, nausea, emotion, and behavior
- mechanism:         Ca2+ increase: Gq activation of PIPLC, giving DAG and IP 3 (Ca2+ channels may contribute)
                     prostaglandin synthesis: Ca2+ activation of PLA2
- regulation:        principal: neuroendocrine reflex and hypothalamic stimulation
                     stimuli: nipples (suckling), GI tract, genital tract (distension of vagina and cervix)
anti-diuretic hormone (ADH, vasopressin)
- function:          increases blood pressure via antidiuretic, vasoconstrictive effects
- synthesis:         single gene product (similar to oxytocin, 9 AA oligopeptide)
- processing:        cleaved into neurophysin II and hormone, transported to axons of the posterior pituitary
- secretion:         released as a hormone-neurophysin II complex
- effects:           antidiuretic: increases H2O reabsorption (increased permeability of distal, collecting tubules)
                     pressor: increases blood pressure (vasoconstriction; minor effect)
- mechanism:         antidiuretic: V2 receptor Gs-coupled increase of cAMP, phosphorylation of aquaporins
                     pressor: V1 receptor Gq-coupled increase of Ca2+ via IP3, DAG  PKC
                     - also promotes glycogen breakdown in liver via PKC)
- regulation:        principal: increased blood osmolarity
                     secondary: large decreases in blood volume (20% or more)
                     others: postural changes, drugs and other stimuli (pain, trauma); inhibited by stress hormones
- clinical:          bed-wetting: may be linked to difficulties in ADH release during sleep
                     diabetes insipidus: dilute polyuria from insufficient ADH production, release, or uptake
                     - insulin and glucagon compensate to make normal glucose (thus non-sweet urine)
                     - levels of ADH can help distinguish between deficiencies in production or uptake
                     - note: high ADH can also be present in diabetes mellitus


Notes: Lecture and Reading
introduction
- central nervous system (CNS): participates in hormonal regulation
- hypothalamus: ultimate integrator of CNS information with respect to the pituitary gland
   - adenohypophysis: regulated through hypothalamic releasing factors
   - neurohypophysis: hypothalamic neural tracts that release hormones directly into the bloodstream
                                                                         Biochemistry: NOTES & OBJECTIVES (page 140 of 165)



hypothalamic releasing factors
- synthesis, release, and effects
   - synthesis:    hypothalamus
   - transport:    hypothalamic-hypophysial portal system of capillaries
   - target:       anterior pituitary hormones
   - function:     act at the level of the anterior pituitary to regulate hormone synthesis, secretion

  TABLE: Hypothalamic Hormones or Factors Controlling the Release of Pituitary Hormones
   full name                                   abbreviation                    hormone secretion affected
   corticotropin-releasing factor              CRF                             ACTH
   (ACTH-releasing factor, corticoliberin)     ACTH-RF
   thyrotropin-releasing hormone               TRH                             TSH, prolactin
   (thyroliberin)
   lutenizing hormone releasing hormone        LHRH                            LH, FSH
   (gonadotropin-releasing hormone, luliberin) GnRH
   somatostatin                                somatoliberin                   GH, TSH, glucagon, insulin,
   (growth hormone release-inhibiting          GHRIH                           gastrin, secretin, others
   hormone)
   growth hormone releasing factor             GH-RF                           GH
   (somatoliberin)
   prolactin release-inhibiting factor         PIF                             prolactin
   (prolactostatin, dopamine)                  dopamine
   prolactin releasing factor                  PRF                             prolactin

   melanocyte-stimulating hormone                    MIF                                 MSH
    release-inhibiting factor (melanostatin)
   melanocyte-stimulating hormone                    MRF                                 MSH
    releasing factor (melanoliberin)

general regulation
- general process
   - releasing factors: secreted from hypothalamus as a result of extrahypothalamic or neural stimuli
   - tropic hormones: release/synthesis in anterior pituitary as a result of releasing factors
   - end organ hormones: final hormones produced in response to tropic hormones; generally feed back
- example: feedback control by the gonads
   - extrahypothalamic stimuli (drugs, stress): stimulate hypothalamus
   - hypothalamus: releases GnRH (releasing factor) into portal system
   - anterior pituitary: releases FSH, LH (tropic hormones) into bloodstream
   - gonads (target organ): produce gonadal steroids (end organ hormones), which cause the metabolic effect
   - gonadal steroids: negatively feed back on anterior pituitary, hypothalamus
general overview of releasing factor mechanisms of action
- chemistry: most are peptides; PIF is dopamine (catecholamine)
- effects: primarily regulate tropic hormone secretion; secondarily regulate synthesis
- receptors: specific receptors for factors on the surface of specific cell types
   - mechanism: changes in intracellular Ca2+, PI metabolism, and/or cAMP
   - specific examples
      - CRF:                  enhances cAMP production in cells
      - TRH, GnRH:            elevate intracellular Ca2+ via production of IP3 (release from ER) and DAG ( PKC)
- hypothalamic integration
   - hypothalamus: organization allows processing of neurogenic signals from many sources
      - changes in internal environment
      - changes in external environment
      - hormonal feedback signals
   - pituitary hormones: levels directly, indirectly modified by hyopthalamus
      - fluctuate wildly in response to diet, activity, stress, sleep, and other important factors
                                                                      Biochemistry: NOTES & OBJECTIVES (page 141 of 165)



      - half lives: 15-20 minutes
   - neurogenic stimuli
      - ACTH:                  circadian, psychogenic, and stress-induced release
      - TSH:                   cold exposure in children; also somewhat in adolescence, adulthood
      - gonadotropins:         regulated by numerous extrahypothalamic signals, including the pineal gland
      - GH, gonadotropins: regulated by sleep-related precesses
posterior pituitary hormones: overview
- introduction
   - posterior pituitary: distal component of a direct neurosecretory system beginning in the hypothalamus
   - major hormones
      - oxytocin: major hormone of ejection (babies, milk, vomit, sexual fluids)
      - ADH (vasopressin): increases blood pressure through vasoconstrictive, anti-diuretic effects
- general chemistry: oxytocin and ADH
   - structure:      oligopeptides (9 AA) in length, differing only at positions 3, 8
   - synthesis:      created as part of a precursor which is then cleaved into the hormone and a neurophysin
                     - neurophysin: precursor remainder, remains non-covalently bound to the oligopeptide hormone
                     - serves as a built-in transport system for the hormone, giving a time delay for bioactivity
oxytocin
- synthesis:         single gene product
- processing:        cleaved into neurophysin I and hormone, transported to axons of the posterior pituitary
- secretion:         released as a hormone-neurophysin I complex
- effects:           females: stimulates contraction of the uterus and the mammary alveoli
                     - permissive effects: requires estrogen priming, lactating conditions
                     males: inhibits androgen biosynthesis in testis (mechanisms, logic are unclear)
                     others: may also have effects on satiety, nausea, emotion, and behavior
- mechanism:         Ca2+ increase: Gq activation of PIPLC, giving DAG and IP 3 (Ca2+ channels may contribute)
                     prostaglandin synthesis: Ca2+ activation of PLA2
- regulation:        principal: neuroendocrine reflex and hypothalamic stimulation
                     stimuli: nipples (suckling), GI tract, genital tract (distension of vagina and cervix)
anti-diuretic hormone (ADH, vasopressin)
- synthesis:         single gene product (similar to oxytocin)
- processing:        cleaved into neurophysin II and hormone, transported to axons of the posterior pituitary
- secretion:         released as a hormone-neurophysin II complex
- effects:           antidiuretic: increases H2O reabsorption (increased permeability of distal, collecting tubules)
                     pressor: increases blood pressure (vasoconstriction; minor effect)
                     other: may also have effects on memory
- mechanism:         antidiuretic: V2 receptor Gs-coupled increase of cAMP, phosphorylation of aquaporins
                     pressor: V1 receptor Gq-coupled increase of Ca2+ via IP3, DAG  PKC (also promotes glycogen
                               breakdown in liver via PKC)
- regulation:        principal: blood osmolarity
                     - released by increased blood osmolarity, diminished by hypotonicity
                     - sensors: osmoreceptors in the hypothalamus
                     secondary: large decreases in blood volume (20% or more)
                     - mediated by baroreceptors in the vasculature
                     - serves as a last-ditch trauma-induced effort to maintain blood pressure
                     others: postural changes, drugs and other stimuli
                     - release activated by pain, trauma
                     - release inhibited by adrenaline, cortisol, or ethanol
- clinical:          bed-wetting: may be linked to difficulties in ADH release during sleep
                     diabetes insipidus: dilute polyuria produced from insufficient ADH production/release
                     - insulin and glucagon compensate to make normal glucose (thus non-sweet urine)
                     - levels of ADH can help distinguish between deficiencies in production or uptake
                     - note: high ADH can also be present in diabetes mellitus
                                                                       Biochemistry: NOTES & OBJECTIVES (page 142 of 165)




59. Water and Electrolyte Regulation
Study Guide
- regulation of renal H2O output
   - electrolyte and water balance: the “A” hormones
      - anti-diuretic hormone (ADH): increases H2O reuptake via collecting tubule aquaporins
      - angiotensin-renin system: causes synthesis, release of aldosterone; angiotensin II is a powerful vasoconstrictor
      - aldosterone: increases Na+ reuptake via synthesis of distal tubule Na + pumps
      - atrial natriuretic factor (ANF): decreases BP by opposing effects of ADH, angiotensin-renin, and aldosterone
   - sodium balance
      - Na+: generally the major osmotically-active substance in the blood (changed in diabetes mellitus)
         - passive: most salt reabsorbed in proximal tubule, loop of Henley
         - active: in distal portion of the kidney, Na+ reabsorbed in a regulated, ATP-dependent manner
      - Na+ regulation: indirectly determines water balance
      - separate mechanisms: Na+, H2O are regulated separately, but are often interconnected
anti-diuretic hormone
- structure:          oligopeptide
- synthesis:          produced in the neurohypophysis
- function:           promotes uptake of H2O in collecting tubules via enhanced expression of aquaporins
- mechanism:          Gs activation of the cAMP-PKA system (via V2 receptors) to phosphorylate aquaporins
                      Gq-mediated Ca2+ increase to stimulate vasoconstriction
- regulation:         stimulation of hypothalamic osmo- and baroreceptors
angiotensin II
- structure:          polypeptide
- synthesis:          cleavage product of angiotensin I (via ACE), which is a product of circulating angiotensinogen
- function:           promotes peripheral vasoconstriction, synthesis of aldosterone
- mechanism:          Gq stimulation of PI PLC, giving DAG and IP 3, which work to increase Ca2+
- regulation          renin: secreted by kidney JG cells in response to decreases in blood pressure, volume, Na + levels
                      renin cleaves circulating angiotensinogen into angiotensin I
                      angiotensin-converting enzyme (ACE) cleaves angiotensin I into angiotensin II
                      angiotensin II can further be cleaved into angiotensin III
aldosterone
- structure:          mineralcorticoid
- synthesis:          produced in the adrenal cortex in response to elevated angiotensin II
- function:           promotes the retention of Na +, excretion of K+ in the distal portion of the kidney
- mechanism:          enters cell, forms nuclear steroid-receptor complex that regulates Na + pump enzymes
- regulation:         renin-angiotensin system: angiotensin II, angiotensin III stimulates aldosterone synthesis
                      Na+, K+: concentrations influence aldosterone synthesis
                      ACTH: permissive/supportive role
atrial natriuretic factor
- structure:          polypeptide hormone
- synthesis:          produced by the heart in response to elevated blood pressure
- function:           works to lower blood pressure (opposes renin-angiotensin II-aldosterone system)
- mechanism:          ANF-R1: coupled to guanylate cyclase ( cGMP), giving ANF’s biological effects
                      ANF-R2: not coupled to guanylate cyclase; work to clear ANF from circulation
- regulation:         produced in response to elevated blood pressure in the heart
- effects:            Na+ uptake: increase GFR, inhibit tubular reabsorption of Na +
                      - inhibits synthesis, release of aldosterone, partially by antagonism of angiotensin III
                      - antagonizes pressor effect of angiotensin II
                      net effect: directly lowers blood pressure, indirectly reduces volume (through Na + regulation)
selected hormonal disease states
- hypoaldosteronism (adrenal cortical insufficiency, Addison’s disease)
   - mechanism: excess salt loss, with osmotic reduction causing deficiency in ADH, resulting in water loss
                      insufficient Na+ reabsorption  insufficient H+, K+ excretion
                                                                     Biochemistry: NOTES & OBJECTIVES (page 143 of 165)



   - result:       metabolic acidosis
   - symptoms: dehydration, high blood pressure, acidosis
- hyperaldosteronism
   - mechanism: excess salt reabsorption, with osmotic excess causing H2O retention and K+ and H+ loss
   - result:       metabolic alkalosis
- diabetes mellitus
   - mechanism: glucosuria: glucose exceeds renal threshold
                   ketonuria: ketone bodies exceed renal threshold
                   polyuria: excess glucose, ketone bodies act as osmotic diuretics, causing polyuria
   - result:       major loss of water, Na+ via osmosis and electroneutrality with ketone bodies
                   ADH: released to combat increased blood osmolarity, loss of body water
                   renin: released to combat Na+ loss, drop in blood volume/pressure
   - symptoms: metabolic disturbances, ketoacidosis, and extensive alterations in water and electrolyte balance


Notes: Lecture and Reading
water balance
- introduction: water intake, output
   - control: nervous, hormonal mechanisms that regulate water intake and loss
   - intake: regulated by diet, neuronal control at the hypothalamus and higher CNS levels
      - metabolic water: available via CHO, protein, and fat oxidation
      - intake generally exceeds body needs, and output is regulated accordingly
   - output: occurs in various ways, including through skin, lungs, GI, and the kidney
      - water balance associated with gain, loss of electrolytes
      - methods of output
         - expired air, evaporation: not under regulatory control
         - renal output: regulated to maintain balance; certain minimal amount (~300 mL) required for waste removal

  TABLE: Water Intake and Output in Typical H2O balance
   intake (mL)                      output (mL)
   drink:         1000              skin:            400
   food:          700               lungs:           400
   metabolism:    300               GI tract:        200
                                    kidney:          1000
   TOTAL:         2000 mL           TOTAL:           2000 mL

- regulation of renal H2O output: overview
   - water balance: “A”
      - anti-diuretic hormone (ADH)
      - angiotensin-renin system
      - aldosterone
      - atrial natriuretic factor (ANF)
   - sodium
      - Na+: generally the major osmotically-active substance in the blood (changed in diabetes mellitus)
      - Na+ regulation: indirectly determines water balance
         - gain in Na+ generally gives a gain in H2O
         - loss in Na+ generally gives a loss in H2O
      - separate mechanisms for control of Na+, H2O losses
         - substances can thus be handled separately: either can be preferentially excreted/retained
         - both mechanisms of control are often simultaneously activated or interconnected
- role of the kidney and ADH
   - filtration
      - renal blood flow:               ~1270 mL/min
      - filtrate production:            130 mL/min, or 187 L/day (hematocrit of 45%, giving 700 mL plasma/min
      - filtrate composition:           plasma minus proteins (composition altered by reabsorption)
      - urine volume:                   0.5% of the glomerular filtrate
                                                                         Biochemistry: NOTES & OBJECTIVES (page 144 of 165)



   - increase in blood volume: mechanisms
      - isoosmotic: passive reabsorption of H2O and most salts (80-87%); not under hormonal control
      - selective: increased by ADH in the distal portion of the tubule, including the collecting ducts
         - ADH released after stimulation of osmo- and baroreceptors
         - mechanism: Gs activation of the cAMP-PKA system (via V2 receptors) to phosphorylate aquaporins
         - function: increase membrane permeability, reuptake of water and electrolytes
         - diabetes insipidus: complete lack of ADH, which can result in daily loss of up to 27 L urine
salt balance, angiotensin, and aldosterone
- salt balance
   - passive: most salt reabsorbed in proximal tubule, loop of Henley
   - active: in distal portion of the kidney, Na+ reabsorbed in a regulated, ATP-dependent manner
      - Na+:                    requires a specific transport protein
      - Cl-, HCO3-:             reabsorbed passively
      - K+, H+:                 excreted isoelectrically as a consequence of Na + absorption
- renin-angiotensin system
   - hormones
      - renin: secreted by kidney JG cells in response to decreases in blood pressure, volume, or Na + levels
      - angiotensinogen: circulating plasma α2 globulin produced by the liver
      - angiotensin I: peptide cleavage product of angiotensinogen; not active
      - angiotensin II: peptide cleavage product of angiotensin I; increases blood pressure
         - most powerful pressor agent known; responsible for essential hypertension
         - also acts on adrenal cortex to stimulate release of aldosterone (increases H 2O reabsorption)
         - mechanism: increased Ca2+ through Gq-linked stimulation of PI PLC (giving DAG, IP 3)
      - angiotensin III: peptide cleavage product of angiotensin II; also active in aldosterone production
   - process
      - JG cells: sense decreased blood pressure, volume, or Na + levels, secrete renin
      - renin cleaves circulating angiotensinogen into angiotensin I
      - angiotensin I is cleaved to angiotensin II
         - enzyme: angiotensinase, angiotensin-converting enzyme, or ACE (primarily in the lung)
         - this enzyme is the target of ACE inhibitors, which are intended to reduce blood pressure
      - angiotensin II causes peripheral vasoconstriction, as well as synthesis of aldosterone
- aldosterone
   - structure:       mineralcorticoid
   - synthesis:       produced in the adrenal cortex
   - function:        promotes the retention of Na +, excretion of K+ in the distal portion of the kidney
   - mechanism: enters cell, forms steroid-receptor complex in the nucleus
                      regulates synthesis of specific transport proteins and/or enzymes necessary for Na + pump
   - regulation: renin-angiotensin system: angiotensin II, angiotensin III stimulates aldosterone synthesis
                      Na+, K+: concentrations influence aldosterone synthesis
                      ACTH: permissive/supportive role
- electrolyte balance: maintained through actions of ADH, aldosterone
atrial natriuretic factor
- atrial natriuretic factor
   - structure:       polypeptide hormone
   - synthesis:       produced in the heart
   - function:        works to lower blood pressure (opposes renin-angiotensin II-aldosterone system)
   - mechanism: ANF-R1: coupled to guanylate cyclase ( cGMP), giving ANF’s biological effects
                      ANF-R2: not coupled to guanylate cyclase; work to clear ANF from circulation
   - effects:         Na+ uptake: increase GFR, inhibit tubular reabsorption of Na +
                      aldosterone: inhibits synthesis, release of aldosterone, partially by antagonism of angiotensin III
                      angiotensin II: antagonizes pressor effect
                      net effect: directly lowers blood pressure, indirectly reduces volume (through Na + regulation)
selected hormonal disease states
- hypoaldosteronism (adrenal cortical insufficiency, Addison’s disease)
   - mechanism: excess salt loss, with osmotic reduction causing deficiency in ADH, resulting in water loss
                      insufficient Na+ reabsorption  insufficient H+, K+ excretion
                                                                     Biochemistry: NOTES & OBJECTIVES (page 145 of 165)



   - result:       metabolic acidosis
   - symptoms: dehydration, high blood pressure, acidosis
- hyperaldosteronism
   - mechanism: excess salt reabsorption, with osmotic excess causing H2O retention and K+ and H+ loss
   - result:       metabolic alkalosis
- diabetes mellitus
   - mechanism: glucosuria: glucose exceeds renal threshold
                   ketonuria: ketone bodies exceed renal threshold
                   polyuria: excess glucose, ketone bodies act as osmotic diuretics, causing polyuria
   - result:       major loss of water, Na+ via electroneutrality with ketone bodies
                   release of ADH to combat increased blood osmolarity, loss of body water
                   release of renin to combat Na+ loss, drop in blood volume/pressure
   - symptoms: metabolic disturbances, ketoacidosis, and extensive alterations in water and electrolyte balance




60., 61. Anterior Pituitary Hormones
Study Guide
adrenocorticotropic hormone (ACTH, corticotropin)
- structure:       straight chain polypeptide, highly conserved in mammalian species
- group:           corticotropin-lipotropin
- effects:         growth and function of the adrenal cortex
                   - stimulates glucocorticoid (cortisol) production and secretion
                   - permissive to aldosterone (mineralcorticoid) secretion and production
- mechanism:       Gs-mediated elevation of cAMP, leading to PKA
                   - stimulates the rate-limiting steroidogenal step, cholesterol  pregnenolone [20-22 lyase]
                   - stimulates the hydrolysis of cholesterol esters
- regulation:      neural: corticotropin releasing factor (CRF)
                   negative feedback: cortisol
                   circadian rhythm
thyroid-stimulating hormone (TSH, thyrotropin)
- group:           glycoprotien
- effects:         growth and function of the thyroid gland
                   - stimulates synthesis, release of the iodinated thyroid hormones
                   - iodinated thyroid hormones: thyroxine and triiodothyronine (T 3 and T4) (hydrophobic due to I)
                   stimulates lipolysis in adipose tissue
- mechanism:       Gs-mediated elevation of cAMP
- regulation:      negative feedback: T3, T4
                   - primary means of regulation of release
                   - feedback occurs mostly at the anterior pituitary, rather than the hypothalamus
                   neural: thyrotropin releasing factor (TRF)
- clinical:        TSH has a long N-terminus, which can lead to autoimmune effects, constant activation
                   can lead to high T3 and T4, fever, goiter, Grave’s disease, high insulin metabolism
follicle-stimulating hormone (FSH)
- group:           glycoprotein
- effects:         stimulates growth and function of the gonads and accessory sex organs, induces steroid synthesis
                   female: enhances follicular maturation
                             increases androgen conversion to estrogens
                             upregulates FSH and LH receptor number in granulosa cells
                             helps initiate oogenesis
                             stimulates secretion of inhibin, which inhibits FSH release
                   male: stimulates spermatogenesis
                             induces synthesis of inhibin, androgen-binding protein (Sertoli cells)
                                                                     Biochemistry: NOTES & OBJECTIVES (page 146 of 165)



                             enhances development of seminiferous tubules
- mechanism:      Gs-mediated increase in cAMP
- regulation:     neural: gonadotropin-releasing hormone (GnRH)
                  estrogens: low levels inhibit, high levels stimulate release
                  inhibin: feeds back to inhibit release only of FSH, not LH
lutenizing hormone (LH, interstitial cell stimulating hormone, ICSH)
- effects         female: with FSH, induces final follicular ripening, ovum release, corpus luteum formation
                             increases steroidogenesis by gonads, making estrogens
                  male: stimulates complete maturation of sperm
                             induces cholesterol to androgen conversion (Leydig cells)
- mechanism:      Gs-mediated increase of cAMP, acting on cholesterol ester hydrolysis and 20-22 lyase
- regulation:     neural: gonadotropin-releasing hormone (GnRH)
                  negative feedback: steroids feed back on anterior pituitary, hypothalamus
                  circadian rhythms
- clinical:       defects in steroidogenesis are even more common than diabetes mellitus
growth hormone (GH, somatotropin)
- structure:      single polypeptide chain (191 amino acids), with high species specificity
- effects:        helps liberate fuels from energy rich tissues to promote growth of muscle, bone, cartilage
                  - stimulates skeletal growth
                       - stimulates chondrogenesis, osteogenesis via insulin-like growth factor I (IGF-I)
                       - increases collagen synthesis, calcification, and growth of the long bones at epiphyses
                       - note: will not enhance linear growth after puberty, but can lead to acromegaly
                  - stimulates general protein synthesis
                       - tissues: muscle, heart, bone, liver
                       - effects: AA uptake, mRNA synthesis, tRNA synthesis, protein synthesis
                  - increases muscle glycogen
                       - muscle: IGF-I mediated activation of glycogen synthesis
                       - liver: direct GH stimulation of glycogen breakdown
                       - note: GH is elevated during certain periods of low glucose, giving effects above
                  - increases plasma FFA
                       - stimulates lipolysis through GH-induced synthesis of hormone-sensitive lipase
- mechanism:      most effects mediated by insulin-like growth factors
                  - IGF-I: polypeptide with insulin-like metabolic effects
                       - mechanism: binds to cell-surface receptors, initiates insulin-like processes
                       - receptor: like insulin receptor, has intrinsic tyrosine kinase (Jak2)
                  - signal transduction
                       - GH binds 2 GH receptors, which activate intracellular tyrosine kinases (Jak2)
                       - Jak2 phosphorylates the “signal transducers and activators of transcription (STATs)
                       - STATs dimerize, enter the nucleus, and modulate transcription
                  carriers: though water soluble, there are binding proteins for both GH and IGF
- regulation:     circadian: sleep-related release, unrelated to hypoglycemia, allowing for growth even in low I/G
                  neural: GRF and GIF (somatostatin), dependent on blood glucose (not primary regulation)
                  endocrine: activated response to T 3, T4, cortisol
- clinical:       imbalances can cause gigantism, acromegaly, and dwarfism
prolactin (PRL, lactogenic hormone, luteotropic hormone, LtH)
- effects:        development of the mammary gland and milk production during lactation
                  - increased casein, lactalbumin, lactose synthetase, triglyceride synthesis
- mechanism:      similar to GH receptor: Jak2-mediated activation of STATs, modulation of transcription
- regulation:     neural: prolactin inhibitory factor (PIF, dopamine), the primary mode of regulation
                  neural: thyrotropin releasing factor (TRH), a moderate activator
                  endocrine: estrogen, giving a permissive effect
                  negative feedback: prolactin, distension increase release of inhibitory factor


Notes: Lecture and Reading
general considerations
                                                                      Biochemistry: NOTES & OBJECTIVES (page 147 of 165)



- overview
   - type:        polypeptides or glycoproteins (all water-soluble)
   - size:        22 (MSH) to 190-200 (GH) amino acids
   - targets:     most: small range of target tissues
                  GH:      broad range of target tissues
  - processes :   metabolic regulation:      GH, ACTH, TSH
                  reproduction:              LH, FSH, prolactin
  - grouping:     structural similarity

- polypeptide hormones (corticotropin-lipotropin group)
   - included hormones: ACTH (adrenocorticotropic hormone)
                               MSH (melanocyte stimulating factor)
                               LPH (lipotropin)
   - structure: polypeptides derived from a common precursor
   - pro-opiocortin (pro-opiomelanocortin, or POMC)
      - pro-opiocortin: gene with extremely complex post-translational processing
      - precursor to several polypeptide hormones that affect stress levels
- glycoprotein hormones
   - included hormones: TSH (thyroid-stimulating hormone)
                               LH (lutenizing hormone, a gonadotropin)
                               FSH (follicle-stimulating hormone, a gonadotropin)
   - structure: glycoproteins (25-30 kDa) consisting of two dissimilar subunits (α, β)
      - subunits
         - α subunit: nearly identical in primary structures, placement of CHO side chains
         - β subunit: more variable structure, conferring structural specificity
      - hybrid hormones
         - separate α, β subunits: little biological activity
         - hybrid hormones: assume the hormonal character of the β subunit
      - carbohydrate modification: variable, Asn-linked, and key to determining half life and potency
- very big hormones
   - included hormones: GH (growth hormone)
                               prolactin
                               placental lactogen
   - structure: single chain of ~191 amino acids, with strong sequence homology

SPECIFIC ANTERIOR PITUITARY HORMONES
- overview
   - regulation
      - largely regulated via the CNS and hypothalamic releasing hormones
      - also regulated by end organ hormones and other substances from non-endocrine tissues
   - categories
      - polypeptide hormones:         corticotropin-lipotropin group; derived from pro-opiocortin
      - glycoprotein hormones:        thyrotropin, gonadotropins
      - others:                       growth hormone, prolactin

corticotropin-lipotropin group
- adrenocorticotropic hormone (ACTH, corticotropin)
   - structure:  straight chain polypeptide, highly conserved in mammalian species
   - effects:    growth and function of the adrenal cortex
                 - stimulates glucocorticoid (cortisol) production and secretion
                 - permissive to aldosterone (mineralcorticoid) secretion and production
   - mechanism: Gs-mediated elevation of cAMP, leading to PKA
                 - stimulates the rate-limiting steroidogenal step, cholesterol  pregnenolone [20-22 lyase]
                 - stimulates the hydrolysis of cholesterol esters
   - regulation: neural: corticotropic releasing factor (CRF)
                 negative feedback: cortisol
                                                                     Biochemistry: NOTES & OBJECTIVES (page 148 of 165)



                   circadian rhythm
- lipotropin (β-LPH)
   - structure:    polypeptide hormone (91 amino acids)
                   - portions of the chain contain AA sequences of opiate-like neural oligopeptides
                   - opiate-like oligopeptides: endorphins, encephalins
   - effects:      increases lipolysis in adipose tissue
- melanocyte-stimulating hormone (MSH)
   - structure:    straight chain polypeptides (α-MSH, β-MSH; no relation to α, β glycoproteins)
   - effects:      dispersal of melanin granules and darkening of skin
                   - α-MSH: general anti-inflammatory activity
                   - excess β-MSH: hyperpigmentation
- encephalins, endorphins
   - structure:    polypeptide hormone
   - effects:      produced centrally during extreme stress, shock, and exercise via pro-opiocortin processing
                   - codeine, morphine, heroin: bind same receptors, mimic actions
glycoprotein hormones
- thyroid-stimulating hormone (TSH, thyrotropin)
   - effects:      growth and function of the thyroid gland
                   - stimulates synthesis, release of the iodinated thyroid hormones
                   - iodinated thyroid hormones: thyroxine and triiodothyronine (T 3 and T4)
                   - iodine, a large molecule, makes T 3 and T4 quite hydrophobic
                   stimulates lipolysis in adipose tissue
   - mechanism: Gs-mediated elevation of cAMP
   - regulation: negative feedback: T3, T4
                   - primary means of regulation of release
                   - feedback occurs mostly at the anterior pituitary, rather than the hypothalamus
                   neural: thyrotropin releasing factor (TRF)
   - clinical:     TSH has a long N-terminus, which can lead to autoimmune effects, constant activation
                   can lead to high T3 and T4, fever, goiter, Grave’s disease, high insulin metabolism
- follicle-stimulating hormone (FSH)
   - effects:      stimulates growth and function of the gonads and accessory sex organs, induces steroid synthesis
                   female: enhances follicular maturation
                             increases androgen conversion to estrogens
                             upregulates FSH and LH receptor number in granulosa cells
                             helps initiate oogenesis
                             stimulates secretion of inhibin, which inhibits FSH release
                   male: stimulates spermatogenesis
                             induces synthesis of androgen-binding protein, inhibin
                             enhances development of seminiferous tubules
   - mechanism: Gs-mediated increase in cAMP
   - regulation: neural: gonadotropin-releasing hormone (GnRH)
                   estrogens: low levels inhibit, high levels stimulate release
                   inhibin: feeds back to inhibit release only of FSH, not LH
- lutenizing hormone (LH, interstitial cell stimulating hormone, ICSH)
   - effects       female: with FSH, induces final follicular ripening, ovum release, corpus luteum formation
                             increases steroidogenesis by gonads, making estrogens
                   male: stimulates complete maturation of sperm
                             induces cholesterol to androgen conversion
   - mechanism: Gs-mediated increase of cAMP, acting on cholesterol ester hydrolysis and 20-22 lyase
   - regulation: neural: gonadotropin-releasing hormone (GnRH)
                   negative feedback: steroids feed back on anterior pituitary, hypothalamus
                   circadian rhythms
   - clinical:     defects in steroidogenesis are even more common than diabetes mellitus
- human chorionic gonadotropin (HCG, a placental gonadotropin)
   - structure:    similar to LH: glycoprotein of an α and β subunit
                   - α subunit: identical to LHα
                                                                      Biochemistry: NOTES & OBJECTIVES (page 149 of 165)



                  - β subunit: larger, contains more carbohydrate
   - clinical:    measurement of β subunit the basis for most pregnancy tests
growth hormone and prolactin
- growth hormone (GH, somatotropin)
   - structure:   single polypeptide chain (191 amino acids)
                  high species specificity: only monkey, human GH actively promote growth in humans
   - effects:     helps liberate fuels from energy rich tissues to promote growth of muscle, bone, cartilage
                  - stimulates skeletal growth
                       - stimulates chondrogenesis, osteogenesis via insulin-like growth factor I (IGF-I)
                       - increases collagen synthesis, calcification, and growth of the long bones at epiphyses
                       - note: will not enhance linear growth after puberty, but can lead to acromegaly
                  - stimulates general protein synthesis
                       - tissues: muscle, heart, bone, liver
                       - effects: AA uptake, mRNA synthesis, tRNA synthesis, protein synthesis
                  - increases muscle glycogen
                       - muscle: IGF-I mediated activation of glycogen synthesis
                       - liver: direct GH stimulation of glycogen breakdown
                       - note: GH is elevated during certain periods of low glucose, giving effects above
                  - increases plasma FFA
                       - stimulates lipolysis through GH-induced synthesis of hormone-sensitive lipase
   - mechanism: most effects mediated by insulin-like growth factors
                  - IGF-I: polypeptide with insulin-like metabolic effects
                       - mechanism: binds to cell-surface receptors, initiates insulin-like processes
                       - receptor: like insulin receptor, has intrinsic tyrosine kinase (Jak2)
                  - signal transduction
                       - GH binds 2 GH receptors, which activate intracellular tyrosine kinases (Jak2)
                       - Jak2 phosphorylates the “signal transducers and activators of transcription (STATs)
                       - STATs dimerize, enter the nucleus, and modulate transcription (IGF-I, HSL)
                  carriers: though water soluble, there are binding proteins for both GH and IGF
   - regulation: circadian: sleep-related release, unrelated to hypoglycemia
                  - glucagon (catabolic) is elevated during sleep
                  - because of this regulation, GH is able to promote anabolism even with low I/G
                  - if muscles had glucagon receptors, growth would be poor
                  neural: GRF and GIF (somatostatin), dependent on blood glucose (not primary regulation)
                  endocrine: response to T3, T4, cortisol
   - clinical:    imbalances can cause gigantism, acromegaly, and dwarfism
- prolactin (PRL, lactogenic hormone, luteotropic hormone, LtH)
   - effects:     development of the mammary gland and milk production during lactation
                  - increased casein, lactalbumin, lactose synthetase, triglyceride synthesis
                  weak GH-like properties, osmoregulation, antigonadotropic, immune functions
   - mechanism: similar to GH receptor: Jak2-mediated activation of STATs, modulation of transcription
   - regulation: neural: prolactin inhibitory factor (PIF, dopamine), the primary mode of regulation
                  neural: thyrotropin releasing factor (TRH), a moderate activator
                  endocrine: estrogen, giving a permissive effect
                  negative feedback: prolactin, distension increase release of inhibitory factor




62., 63. Steroid Hormone Biosynthesis and Regulation
Study Guide
- three important steroids of the adrenal cortex
   - aldosterone
      - classification:     mineralcorticoid
                                                                         Biochemistry: NOTES & OBJECTIVES (page 150 of 165)



     - localization:         zona glomerulosa (outermost layer)
  - cortisol
     - classification:       glucocorticoid (principal glucocorticoid)
     - localization:         zona fasciculata, zona reticularis
  - corticosterone
     - classification:       glucocorticoid (weak glucocorticoid)
     - localization:         zona fasciculata, zona reticularis

overview of adrenal steroidogenesis
- regulated steps
   - 20-22 lyase:         cholesterol  pregnenolone [ACTH is permissive, reaction activated by angiotensin II]
   - 18 hydroxylase DH: corticosterone  aldosterone [activated by angiotensin II]
- horizontal pathways
   - DHEA pathway
      - 17α-hydroxylase:                   pregnenolone  17α-OH-pregnenolone
      - 17-20 lyase:                       17α-OH-pregnenolone  dehydroepiandrosterone (DHEA)
   - androstenedione pathway
      - 17α-hydroxylase:                   progesterone  17α-OH-progesterone
      - 17-20 lyase:                       17α-OH-progesterone  androstenedione

- vertical pathways
   - aldosterone pathway
      - 3β-hydroxysteroid DH/isomerase:        pregnenolone  progesterone
      - 21-hydroxylase:                        progesterone  dehydroxycorticosterone (MC)
      - 11β-hydroxylase:                       dehydroxycorticosterone  corticosterone (GC)
      - 18-hydroxylase DH:                     corticosterone  aldosterone (MC)
                                               - activated by angiotensin II
  - cortisol pathway
     - 3β-hydroxysteroid DH/isomerase:         17α-OH-pregnenolone  17α-OH-progesterone
     - 21-hydroxylase:                         17α-progesterone  dexoycortisol
     - 11β-hydroxylase:                        cortisol (GC)

  - adrenal androgen path:                     dehydroepandrosterone (DHEA, C19)  androstenedione (C19)
                                               androstenedione  testosterone
                                               testosterone  estradiol (trace)
- synthetic tendencies
   - adrenal cortex: makes weak androgens through both pregenolone and progesterone (and thus higher DHEA)
   - gonads: make androgens through progesterone (and thus very little DHEA
aldosterone (mineralcorticoids)
- function:        regulation of Na+ intake to maintain electrolyte and fluid balance
- synthesis:       zona glumerulosa of the adrenal gland
- effects:         distal renal tubule: increases reabsorption of Na+, excretion of K+ (enzyme synthesis)
                   skin, muscle, bone: decreases [Na+], increases [K+] in saliva, sweat, and GI contents
- mechanism:       alteration of synthesis (steroid-intracellular receptor complex)
- regulation:      permissive: ACTH (increased precursor synthesis; does NOT feed back on ACTH)
                   metabolite: angiotensin II, III, Na+, K+
                   - angiotensin II: enhances 22-lyase, 18 hydroxylase/DH (Gq release of Ca2+, activation of PKC)
cortisol (glucocorticoids)
- function:        energy metabolism, especially during fasting, CHO deprivation, and stress
- effects:         stimulates gluconeogenesis
                   - inhibits protein synthesis, inhibits peripheral AA uptake and activates liver AA uptake
                   - stimulates liver synthesis of gluconeogenic enzymes, esp. PEPCK
                   - exerts permissive effect on gluconeogenesis induced by glucagon, epinephrine
                   - high (pharmacological) doses: increased glucose, G6P, and thus glycogen storage in liver
                   permissive effect on lipolysis (↑ hormone-sensitive lipase, or HSL)
                   permissive effect on glycogenolysis (glucagon, adrenaline)
                                                                        Biochemistry: NOTES & OBJECTIVES (page 151 of 165)



                   stimulates methylation of norepinephrine to epinephrine (↑ PNMT)
                   - PNMT: phenylethanolamine N-methyltransferase
                   - important enzyme in the adrenergic response
                   anti-inflammatory effect
- mechanism:       transcriptional: modulation of gene transcription (intracellular receptor-steroid complex)
                   post-transcriptional: mRNA stability
                   permissive: increased synthesis of protein kinase, downstream enzymes (e.g. HSL)
- regulation:      feedback controls: negative feedback on CRF, ACTH levels
                   circadian rhythms: highest in the morning, lowest before bedtime
                   stress: (trauma, infection) increases cortisol production
                   ACTH: stimulates glucocorticoid synthesis (activation of 20-22 lyase)
                   transport: bound to albumin, cortisol-binding globulin (CBG)
                   - CBG: increased by estrogens, thyroid hormones; decreased by nephrosis
                   - CBG: also transports corticosterone and progesterone; not aldosterone
                   metabolism: primarily sulfonation in the liver, increasing solubility and excretion
selected disorders of the adrenal cortex
- adrenocortical insufficiency
   - cause:        decreased glucocorticoid and/or mineralcorticoid production
   - effects:      primary (Addison’s disease): defective production of adrenocorticoids, leading to high ACTH
                   secondary: defective production of ACTH, leading to low adrenocorticoids
- excessive adrenocorticoid levels (Cushing’s syndrome)
   - cause:        tumors of the adrenal cortex
                   excessive pituitary/ectopic production of ACTH
   - effects:      elevated cortisol: muscle wasting, central deposition of body fat, elevated glucose (anti-insulin)
                   elevated weak androgens: masculinization and acne
                   elevated DOC: Na+ retention, water retention leading to hypertension
- congenital adrenal hyperplasias
   - cause:        deficiency or impairment in one or more of the enzymes of adrenal steroid synthesis
   - effects:      accumulation, increased release of steroids formed prior to the defect
                   - effect can be enhanced by compensatory ACTH secretion, lack of cortisol feedback

  TABLE: Presentation: Congenital Adrenal Hyperplasias
                     21 hydroxylase        11β-hyrdoxylase                17α-hydroxylase          18-hydroxylase /DH
   cortisol          low                   low                            low                      normal
   ACTH              elevated              elevated                       elevated                 normal
   DHEA              elevated              elevated                       low                      normal
   DOC               low                   elevated                       elevated                 normal
   aldosterone       low                   low                            low (lack of AII)        low
   [Na+]             low                   elevated                       elevated                 low
   blood pressure    low                   elevated                       elevated                 low



Notes: Lecture and Reading
introduction: the adrenal cortex
- classification by physiological activity (relative; some overlap)
   - mineralcorticoids
   - glucocorticoids
- three important steroids of the adrenal cortex
   - aldosterone
      - classification:     mineralcorticoid
      - localization:       zona glomerulosa (outermost layer)
   - cortisol
      - classification:     glucocorticoid (principal glucocorticoid)
      - localization:       zona fasciculata, zona reticularis
                                                                         Biochemistry: NOTES & OBJECTIVES (page 152 of 165)



   - corticosterone
      - classification:        glucocorticoid (weak glucocorticoid)
      - localization:          zona fasciculata, zona reticularis
chemistry of the corticosteroids
- stereochemistry
   - core steroid structure: cyclopentanoperhydrophenanthrene ring system
   - chiral centers:           carbon atoms shared by two rings must be specified
                               carbons with H that have been replaced also must be specified
- configuration
   - α-substituents:           groups lying below the plane of the drawn ring structure
   - β-substituents:           groups lying above the plane of the drawn ring structure
- nomenclature
   - numbering
      - rings:                 A, B, C, D (left to right)
      - numbers:               1-10: loop around A, B
                               11-17: figure 8 around C, D
                               18:       β-methyl group off 13
                               19:       β-methyl group off 10
                               20, 21: first C of tail off β-17; methyl group
                               22-26: linear chain off 20
                               27:       methy groups off 25
   - stereochemical configuration
      - C19 (methyl group off C10) determines α or β steroid
      - C5, if it does not have a double bond, stereochemistry of H should be specified
   - nomenclature
      - gonane:                C17       core of structures of the gonads
      - estrane:               C18       core of estrogens
      - androstane:            C19       core of androgens
      - pregnane:              C21       core of progestins: progesterone, aldosterone, cortisol
      - cholane:               C24       core of bile acids
      - cholestane:            C27       cholesterol
   - substituents
      - unsaturation:          -ane to -ene, -diene, -triene…         (preceeded by appropriate numbers)
      - hydroxyl group:        to -ol, -diol, -triol…                 (preceeded by appropriate numbers)
      - carbonyl group:        to -one, -dione, -trione…              (preceeded by appropriate numbers)
overview of adrenal steroidogenesis
- principal regulated step
   - 20-22 lyase:              cholesterol  pregnenolone
                               - ACTH is permissive
                               - reaction activated by angiotensin II]
- horizontal pathways
   - DHEA pathway
      - 17α-hydroxylase: pregnenolone  17α-OH-pregnenolone
      - 17-20 lyase:           17α-OH-pregnenolone  dehydroepiandrosterone (DHEA)
   - androstenedione pathway
      - 17α-hydroxylase: progesterone  17α-OH-progesterone
      - 17-20 lyase:           17α-OH-progesterone  androstenedione
- vertical pathways
   - aldosterone pathway
      - 3β-hydroxysteroid DH/isomerase:             pregnenolone  progesterone
      - 21-hydroxylase:                             progesterone  dehydroxycorticosterone (MC)
      - 11β-hydroxylase:                            dehydroxycorticosterone  corticosterone (GC)
      - 18-hydroxylase DH:                          corticosterone  aldosterone (MC)
                                                    - activated by angiotensin II
   - cortisol pathway
      - 3β-hydroxysteroid DH/isomerase:             17α-OH-pregnenolone  17α-OH-progesterone
                                                                      Biochemistry: NOTES & OBJECTIVES (page 153 of 165)



    - 21-hydroxylase:                        17α-progesterone  dexoycortisol
    - 11β-hydroxylase:                       cortisol (GC)

  - adrenal androgen path:                   dehydroepandrosterone (DHEA, C19)  androstenedione (C19)
                                             androstenedione  testosterone
                                             testosterone  estradiol (trace)
- enzymes
   - horizontal
      - 20-22 lyase: principal regulated step (activated by angiotensin II, with permissive effect by ACTH)
      - 17α-hydroxylase: from aldosterone pathway to cortisol pathway
      - 17-20 lyase: from cortisol pathway to androgen pathway
   - vertical
      - 3β-hydroxysteroid DH/isomerase: first step in aldosterone, cortisol pathways
      - 21-hydroxylase: commits to either aldosterone or cortisol pathway; directly makes cortisol
      - 18-hydroxylase DH: leads to aldosterone
- synthetic tendencies
   - adrenal cortex
      - weak androgens: makes weak androgens through both pregnenolone, progesterone
      - tumor of adrenal cortex: excessive DHEA, excess testosterone
   - gonads
      - weak androgens: tends to use progesterone to make androgens
      - tumor of gonads: excessive testosterone, very little DHEA
biological and metabolic effects of the adrenal cortical steroid hormones
- mineralcorticoids
   - principal:     aldosterone
   - function:      electrolyte and fluid regulation
   - effects:       distal renal tubule: increases reabsorption of Na+, excretion of K+
                    - mechanism: induction of Na+ pump via intracellular receptor
                    skin, muscle, bone: decreases [Na+], increases [K+] in saliva, sweat, and GI contents
- glucocorticoids
   - principal:     cortisol (hydrocortisone)
   - function:      energy metabolism, especially during fasting, CHO deprivation, and stress
   - effects:       stimulates gluconeogenesis
                    - inhibits protein synthesis, increasing the pool of AA available
                    - inhibits AA uptake in peripheral tissues
                    - stimulates AA uptake in liver
                    - stimulates liver synthesis of gluconeogenic enzymes, esp. PEPCK
                    - exerts permissive effect on gluconeogenesis induced by glucagon, epinephrine
                    - high (pharmacological) doses: increased glucose, G6P, and thus glycogen storage in liver
                    permissive effect on lipolysis (↑ hormone-sensitive lipase, HSL)
                    permissive effect on glycogenolysis (glucagon, adrenaline)
                    stimulates methylation of norepinephrine to epinephrine (↑ PNMT)
                    - PNMT: phenylethanolamine N-methyltransferase
                    - important enzyme in the adrenergic response
                    anti-inflammatory effect
                    others: erythropoiesis, ↑ gastric acid/pepsinogen, immunosupressants, cardiovascular effects
   - mechanism: transcriptional: modulation of gene transcription in steroid fashion
                    post-transcriptional: mRNA stability
                    permissive: increased synthesis of protein kinase, downstream enzymes (e.g. HSL)

TABLE: Biological Activities (Relative to Cortisone) of Natural Corticosteroids in Adrenalectomized Rats
 steroid                   CHO activity                   Na+ retention                 anti-inflammatory activity
 cortisone                 100                            100                           100
 cortisol                  155                            150                           1,250
 deoxycorticosterone       0                              3,000                         0
                                                                      Biochemistry: NOTES & OBJECTIVES (page 154 of 165)



 aldosterone                  0                             60,000                         0

regulation of the adrenal cortical steroid hormones
- glucocorticoids (cortisol)
   - feedback controls: negative feedback on CRF, ACTH levels
   - circadian rhythms: highest in the morning, lowest before bedtime
   - stress: (trauma, infection) increases cortisol production
   - ACTH: stimulates glucocorticoid synthesis (activation of 20-22 lyase)
   - storage: small amounts stored
   - transport: bound to albumin, cortisol-binding globulin (CBG)
      - CBG: increased by estrogens, thyroid hormones; decreased by nephrosis
      - CBG: also transports corticosterone and progesterone; not aldosterone
- aldosterone
   - localization: synthesized in zona glomerulosa
      - lacks 17α-hydroxylase
      - contains 18 hydroxylase/DH
   - permissive control: ACTH via increased synthesis of precursors (aldosterone does NOT feed back on ACTH)
   - metabolite controls: angiotensin II, angiotensin III, Na +, K+
      - angiotensin II: enhances early (22-lyase) as well as late (18 hydroxylase/DH) stages
      - angiotensin II mechanism: Gq-mediated release of Ca2+, activation of PKC
metabolism of the adrenal cortical steroids
- sites of metabolism: 11-OH, 20-keto, and unsaturated ketone (3-keto-4-ene) moieties
- types of reactions
   - oxidation/reduction
   - hydroxylation
   - hydrogenation of the C4-C5 double bond
   - conjugation to sulfates, glucosiduronates (more water-soluble, pass readily into the urine)
other active steroids secreted from the adrenal cortex
- weak androgens (adrenal androgens): dehydroepiandrosterone (DHEA), androstenedione
- progesterone (small amounts)
- estrogen (trace amounts)
selected disorders of the adrenal cortex
- adrenocortical insufficiency
   - cause:         decreased glucocorticoid and/or mineralcorticoid production
   - effects:       primary (Addison’s disease): defective production of adrenocorticoids, leading to high ACTH
                    secondary: defective production of ACTH, leading to low adrenocorticoids
- excessive adrenocorticoid levels (Cushing’s syndrome)
   - cause:         tumors of the adrenal cortex
                    excessive pituitary/ectopic production of ACTH
   - effects:       elevated cortisol
                    - muscle wasting, central deposition of body fat
                    - elevated blood glucose (anti-insulin effects), correspondingly resulting in elevated insulin
                    elevated weak androgens: masculinization and acne
                    elevated deoxycortisone (mineralcorticoid): Na+ retention, water retention leading to hypertension
- congenital adrenal hyperplasias
   - cause:         deficiency or impairment in one or more of the enzymes of adrenal steroid synthesis
   - effects:       accumulation, increased release of steroids formed prior to the defect
                    - effect can be enhanced by compensatory ACTH secretion, lack of cortisol feedback

- 21-hydroxylase deficiency
   - hormonal:   decreased aldosterone, cortisol production
   - effects:    high ACTH (due to low cortisol), high levels of progesterone and adrenal androgens
   - symptoms: virulization (excess androgens)
                 hyponatremia (lack of mineralcorticoids) and dehydration
                 hypotension
- 11β-hyrdoxylase deficiency
                                                                      Biochemistry: NOTES & OBJECTIVES (page 155 of 165)



  - hormonal:     decreased aldosterone, cortisol production
  - effects:      high ACTH, high levels of deoxycorticosterone (DOC) and deoxycortisol, high adrenal androgens
  - symptoms:     virulization (excess androgens)
                  hypernatremia (DOC, at high levels, acts as a mineralcorticoid)
                  hypertension
- 17α-hydroxylase deficiency
   - hormonal:    decreased cortisol, androgen production; usually also affects the gonads
   - effects:     high ACTH, high levels of deoxycorticosterone (DOC), and high corticosterone
                  low aldosterone (DOC  salt retention, giving low angiotensin II, giving low aldosterone synth.)
   - symptoms: hypernatreamia (DOC, at high levels, acts as a mineralcorticoid)
                  hypertension
                  female phenotype at birth regardless of genetic sex
- 18 hydroxylase/DH deficiency
   - hormonal:    low aldosterone; normal cortisol, ACTH, and androgens
   - effects:     poor upstream regulation of steroidogenesis
   - symptoms: hyponatremia (low DOC from generally poor steroidogenesis), dehydration
                  hypotension
- 3β-hydroxysteroid DH: usually fatal at an early age
- 20-22 lyase: usually fatal at an early age

  TABLE: Presentation: Congenital Adrenal Hyperplasias
                     21 hydroxylase        11β-hyrdoxylase               17α-hydroxylase         18-hydroxylase /DH
   cortisol          low                   low                           low                     normal
   ACTH              elevated              elevated                      elevated                normal
   DHEA              elevated              elevated                      low                     normal
   DOC               low                   elevated                      elevated                normal
   aldosterone       low                   low                           low (lack of AII)       low
       +
   [Na ]             low                   elevated                      elevated                low
   blood pressure    low                   elevated                      elevated                low




64. Gonadal Hormones (Steroidal Sex Hormones)
Study Guide
- gonadal steroids: overview
   - transport:   bind specific plasma transport proteins (modulate levels of free enzyme)
   - binding:     pass through cell membrane, bind internal receptor
   - mechanism: steroid receptor-hormone complex triggers cellular effects (typically at the transcriptional level)
   - metabolism: principal clearance: sulfate conjugation in the liver, making it more readily excreted in urine
progesterone
- function:       prepares endometrium for pregnancy (predominant in the second menstrual phase)
- localization:   corpus luteum, also somewhat in the adrenal cortex
- synthesis:      cholesterol  [20-22 lyase] pregnenolone  progesterone
- metabolism:     adrenals, gonads: precursor to other steroid hormones
                  target tissues: hydrogenation to modulate (activation or inactivation)
                  liver: conjugation with sulfate or glucuronic acid
- effects:        progestational: prepares endometrium for pregnancy; necessary for its continuation
                  lactation: contributes to mammary growth, letdown
                  gonadotropin regulation: anti-ovulatory effect
- mechanism:      general steroid transcriptional model; receptors in uterus, vagina, breast, pituitary, CNS, others
- regulation:     negative feedback: progestin feedback on GnRH, LH
                  transport: carried on cortisol-binding globulin (CBG), albumin
androgens
                                                                      Biochemistry: NOTES & OBJECTIVES (page 156 of 165)



- function:       secondary sex characteristics in males
- localization:   chiefly in the Leydig cells (interstitial cells of the testis); somewhat by the adrenal cortex
- chemistry:      dehydroepiandrosterone (DHEA): principal androgenic hormone of the adrenals
                  androstenedione: interconverts with testosterone; synthesized by both testis and adrenals
                  testosterone: principal androgenic hormone of the testis
                  dihydrotestosterone (DHT): product at target tissues; most potent endogenous androgen
- synthesis:      preferred pathway: progesterone  17α-OH-progesterone  androstenedione  testosterone
                  5α-reductase: testosterone  5α-dihydrotestosterone (DHT)
                  - this step, localized in target tissues, is crucial to obtaining a full androgenic response
                  - deficiencies can cause impaired development of male genitalia
- metabolism:     liver: conjugation, inactivation
                  target tissues: 5α-reductase conversion to DHT
- effects:        development of male secondary sex characteristics
                  anabolic effects: increase in bone, muscle growth through protein synthesis
                  metabolic effects: promotes male reproductive development (provides fructose for sperm)
- mechanism:      general steroid transcriptional model (testosterone, DHT are the hormonally active steroids
- regulation:     negative feedback: testosterone inhibits GnRH, LH secretion
                  transport: carried on sex hormone binding protein, albumin
estrogens
- function:       development and maintenance of the female reproduction tract; secondary sex characteristics
- localization:   chiefly in the ovaries and placenta
- chemistry:      estrone: interconvertible with estradiol; less potent by a factor of 10
                  17β-estradiol: most potent estrogen; by itself, the metabolically active hormone
                  estriol: generally a product of estradiol metabolism except during pregnancy; less potent
- synthesis:      androgens  estrogens [aromization]
                  - androstenedione  estrone
                  - testosterone  17β-estradiol
                  estrone  17β-estradiol
                  17β-estradiol  estrione [16α-hydroxylase; another pathway opens up during pregnancy]
- metabolism:     liver: glucuronic/sulfuronic conjugation and inactivation, as well as by other mechanisms
- effects:        development of female secondary sex characteristics
                  metabolic effects
                  - promotes female reproductive development
                  - increased fat deposition (decreased levels in blood, increased levels in peripheral tissues)
                  - increased protein synthesis (small compared to anabolic steroids)
                  - Ca2+ bone deposition (antagonizes parathyroid hormone); closure of bone epiphyses
                  modulate FSH, LH secretion
- mechanism:      binding of intracellular α, β receptors, interaction with estrogen response elements (EREs)
                  - no consensus on what constitutes an estrogen response element
                  - note: only the 17β-estradiol exhibits receptor-binding effects
- regulation:     negative feedback: gonadotropins feed back on the hypothalamus, pituitary
                  transport: specific carrier proteins, albumin


Notes: Lecture and Reading
introduction and review
- functions of the gonads
   - gamete production
   - hormone synthesis
- gonadotropins (FSH, LH)
   - stimulate, regulate gonadal differentiation and development
   - lead to maturation of germ cells, secretion of gonadal steroid hormones
- gonadal steroid hormones
   - responsible for the growth, maintenance of the accessory sex structures
      - female: fallopian tubes, uterus, vaginal epithelium, mammary glands
      - male: gonadal ducts, seminal vesicles, prostate
                                                                          Biochemistry: NOTES & OBJECTIVES (page 157 of 165)



   - additional effects
      - metabolic effects on muscle, adipose tissue
      - effects on hair distribution, voice, sexual behavior
   - regulation: in the female, cyclical fashion produces conditions suitable for conception
gonadal steroids
- synthesis
   - process: cholesterol  progesterone  androgens  estrogens
   - males and females produce all of these, but in differing amounts

  TABLE: Synthesis and Secretion of Gonadal Steroids
                                    male                                            female
   cholesterol

    progesterone                          little secreted: primarily an             secreted in large amounts (cyclical)
                                          intermediate in synthesis
    androgens (testosterone)              secreted in large amounts                 little secreted: primarily an
                                                                                    intermediate in synthesis
    estrogens (estradiol, estrone)                                                  secreted in large amounts (cyclical)


transport, binding, mode of action, and metabolism of gonadal steroid hormones
- transport:      bind specific plasma transport proteins
                  - carriers can modulate levels of free enzyme
                  - only free enzyme can interact with the target cell
- binding:        pass through cell membrane, bind internal receptor
- mechanism:      steroid receptor-hormone complex triggers cellular effects
                  - typically, this is at the transcriptional level with nuclear receptors
- metabolism:     principal clearance: sulfate conjugation
                  localization: liver, target tissues
                  mechanism: modification of the functional moieties of the steroid hormone

progesterone
- function:        prepares endometrium for pregnancy (predominant in the second menstrual phase)
- localization:    corpus luteum
- chemistry:       cholesterol  [20-22 lyase] pregnenolone  progesterone
- metabolism:      adrenals, gonads: precursor to other steroid hormones
                   target tissues: hydrogenation to modulate (activation or inactivation)
                   liver: conjugation with sulfate or glucuronic acid
- effects:         progestational: prepares endometrium for pregnancy; necessary for its continuation
                   lactation: contributes to mammary growth, letdown
                   gonadotropin regulation: anti-ovulatory effect
                   thermogenic: increases basal temperature during latter half of cycle
                   mineralcorticoid: only in high concentrations
                   estrogen antagonist
                   protein catabolic effect: only in absence of pregnancy
                   hematopoiesis
                   CNS effects: behavior, sleep, temperature regulation, neuronal excitability
                   anesthetic effect
                   immunosupressive agent: only in high concentrations
- mechanism:       general steroid transcriptional model
                   - receptors present in the nucleus of the uterus, vagina, breast, pituitary, brain, others
                   - progesterone is the active form in the uterus; in the CNS, other metabolites as well
- regulation:      negative feedback: progestin feedback on GnRH, LH
                   transport: carried on cortisol-binding globulin (CBG), albumin
- clinical:        progesterone is inactive orally
                                                                      Biochemistry: NOTES & OBJECTIVES (page 158 of 165)



                  synthetic agents have been developed and used in oral contraceptive formulas
androgens
- function:       secondary sex characteristics in males
- localization:   chiefly in the Leydig cells (interstitial cells of the testis)
- chemistry:      testosterone: principal androgenic hormone of the testis
                  androstenedione: interconverts with testosterone; synthesized by both testis and adrenals
                  dehydroepiandrosterone (DHEA): principal androgenic hormone of the adrenals
                  5α-dihydrotestosterone (DHT): product at target tissues; most potent endogenous androgen
- synthesis:      preferred pathway: progesterone  17α-OH-progesterone  androstenedione  testosterone
                  5α-reductase: testosterone  5α-dihydrotestosterone (DHT)
                  - this step, localized in target tissues, is crucial to obtaining a full androgenic response
                  - deficiencies can cause impaired development of male genitalia
- metabolism:     liver: conjugation, inactivation
                  target tissues: 5α-reductase conversion to DHT
- effects:        development of male secondary sex characteristics
                  anabolic effects: increase in bone, muscle growth through protein synthesis
                  metabolic effects: promotes male reproductive development
                  - fructose is the only sugar available to provide energy for sperm motility
                  - testosterone, DHT act by increasing enzymes in a fructose synthesis pathway
- mechanism:      general steroid transcriptional model
                  - DHT or testosterone appear to be the hormonally active steroids
- regulation:     negative feedback: testosterone inhibits GnRH, LH secretion (in excess: gonadal atrophy)
                  transport: carried on sex hormone binding protein, albumin
- clinical:       testosterone is relatively inactive orally
                  synthetic agents have been developed that are more active orally
estrogens
- function:       development and maintenance of the female reproduction tract; secondary sex characteristics
- localization:   chiefly in the ovaries and placenta
- chemistry:      17β-estradiol: most potent estrogen; by itself, the metabolically active hormone
                  estrone: interconvertible with estradiol; less potent by a factor of 10
                  estriol: generally a product of estradiol metabolism except during pregnancy; less potent
- synthesis:      androgens  estrogens [aromization]
                  - androstenedione  estrone
                  - testosterone  17β-estradiol
                  estrone  17β-estradiol
                  17β-estradiol  estrione [16α-hydroxylase; another pathway opens up during pregnancy]
                  note: estrogens are often formed via aromization in the periphery
                  - menstruating women: 40%
                  - postmenopausal women: 100%
                  - men: 85%
- metabolism:     liver: glucuronic/sulfuronic conjugation and inactivation, as well as by other mechanisms
- effects:        development of female secondary sex characteristics
                  metabolic effects
                  - promotes female reproductive development
                  - increased fat deposition (decreased levels in blood, increased levels in peripheral tissues)
                  - increased protein synthesis (small compared to anabolic steroids)
                  - Ca2+ bone deposition (antagonizes parathyroid hormone); closure of bone epiphyses
                  modulate FSH, LH secretion
                  antagonize androgens
                  promote clotting: synthesis of blood-clotting proteins
                  lower cholesterol: high doses
                  increase levels of sex hormone, thyroid hormone plasma binding proteins
- mechanism:      binding of intracellular α, β receptors, interaction with estrogen response elements (EREs)
                  - no consensus on what constitutes an estrogen response element
                  - note: only the 17β-estradiol exhibits receptor-binding effects
- regulation:     negative feedback: gonadotropins feed back on the hypothalamus, pituitary
                                                                     Biochemistry: NOTES & OBJECTIVES (page 159 of 165)



                 transport: specific carrier proteins, albumin
- clinical:      estrogen is relatively inactive orally
                 synthetic agents have been developed that are more active orally




65. Calcitonin, Parathyroid Hormone, and Cholecalciferol:
        Ca2+ Regulation
Study Guide
calcitonin (CT, thyrocalcitonin)
- function:        reduce blood Ca2+ levels (“tone down that calcium!”)
- structure:       single-chain polypeptide (32 amino acids)
- synthesis:       produced by the C cells of the thyroid
- regulation:      serum [Ca2+]: increased blood Ca2+ gives increased secretion
- effects:         bone: increased deposition, decreased release (targets osteoclasts)
                   kidney: increases excretion of Ca2+ and Pi, decreases reabsorption
- mechanism:       Gs-linked elevation of cAMP in target cells
- clinical:        no known associated human disease with deficiency or excess, suggesting compensatory factors
parathormone (parathyroid hormone, PTH)
- function:        increase blood Ca2+ levels (nothing cute here; simply opposes calcitonin)
- structure:       single chain polypeptide (84 amino acids)
- synthesis:       produced by the parathyroid gland; precursor is processed, stored in granules
- regulation:      serum [Ca2+] levels: decreased blood Ca2+ gives increased secretion
- effects:         bone: stimulation of osteolysis, inhibition of collagen synthesis, causing Ca 2+ release
                   kidney: increases Ca2+ in distal tuble; decreases phosphate reabsorption
                   - by mass action, phosphate is drawn from hydroxyapatite in bone, releasing Ca 2+
                   - also activates 1α-hydroxylase activity, leading to production of 1,25-dihydrocholecalciferol
                   - 1,25-dihydrocholecalciferol: active D3 hormone form that promotes intestinal Ca2+ reabsorption
- mechanism:       Gs-linked elevation of cAMP in target cells (targets different cells than calcitonin)
                   Gq-linked phosphoinositide hydrolysis
- clinical:        hypoparathyroidism: rare; usually follows certain surgeries
                   hyperparathyroidism: can proceed from hyperplasia, cancer, or iatrogenic causes
cholecalciferol (Vitamin D3)
- general considerations
   - structure:    steroid-like
   - synthesis:    can be synthesized from cholesterol derivatives (and is not a vitamin in the strict sense)
                   - cholecalciferol: D3; form found in humans, derived from animal products
                   - ergocalciferol: D2; form found in plants
   - metabolism: 7-dehydrocholesterol  cholecalciferol (D3) [UV irradiation]
                   - 7-dehydrocholesterol: intermediate in cholesterol synthesis
                   - cholecalciferol: precursor to active form in humans
   - clinical:     rickets: disease caused by lack of active vitamin D (osteomalacia: adult form)
                   - synthesis from cholesterol is the primary source in an non-supplemented diet
                   - now rare, but used to be prevalent in areas with long winters and poor diet
- cholecalciferol as a hormone or prohormone
   - function:     proper mineralization of bone, maintenance of adequate plasma Ca 2+
   - synthesis:    7-dehydrocholesterol  cholecalciferol (D3) [UV irradiation]
                   cholecalciferol  25-hydroxycholecalciferol (major precursor) [25-hydroxylase: liver]
                   in presence of low Ca2+:
                   25-hydroxycholecalciferol  1α,25-dihydroxycholecalciferol [1-hydroxylase: kidney]
                             - most active form of vitamin D3
                   1α,25-DHCC  1α,24,25-trihydroxycholecalciferol [24-hydroxylase: kidney]
                             (1,24,25-THCC: less active form of vitamin D3)
                                                                    Biochemistry: NOTES & OBJECTIVES (page 160 of 165)



                in presence of normal or high Ca2+:
                25-hydroxycholecalciferol  24,25-dihydroxycholecalciferol [1-hydroxylase: kidney]
                          (24,25-DHCC: inactive form of vitamin D3)
                24,25-DHCC  1α,24,25-trihydroxycholecalciferol [24-hydroxylase: kidney]
                          (1,24,25-THCC: less active form of vitamin D3)
  - regulation: PTH, low Ca2+: enhances expression of 1α-hydroxylase (diverts to active vitamin D)
                high Ca2+: diverts to inactive, less active forms of vitamin D
  - effects:    general steroid transcriptional model
  - targets:    intestine: stimulates Ca2+ absorption
                bone: mobilization of Ca2+, which raises [Ca2+], stimulating resynthesis by osteoblasts
                kidney: along with PTH, increases renal absorption of Ca 2+
                others: possible link to immune system function
  - metabolism: not readily excreted; in low Ca2+, converted to 1,24,25-THCC, which only affects intestine

TABLE: Summary of Hormonal Control of [Ca2+] and Phosphate
 actions      calcitonin                     parathyroid hormone                    1,25-DHCC
 serum [Ca2+] ↓                              ↑                                      ↑
 serum [Pi]   ↓                              ↓                                      ↑
 kidney       ↓ Ca2+ reabsorption            ↑ reabsorption of Ca2+                 ↑ Ca2+ reabsorption
              ↓ Pi reabsorption              ↓ reabsorption of Pi                   ↑ Pi reabsorption
                                             ↑ 1α-hydroxylase activity
 bone         ↓ resorption of bone           ↑ resorption of bone                   Ca2+ mobilization from bone
 GI tract     no effect                      ↑ absorption of Ca2+, Pi               ↑ absorption of Ca2+, Pi
                                             (indirect: increased production
                                             of 1,25-DHCC)


Notes: Lecture and Reading
introduction: calcium regulation
- body Ca2+ distribution
   - total calcium
      - circulation, non-skeletal tissues:      1%
      - bone:                                   99 % (constitutes a mass of ~ 1kg in humans)
   - bone composition
      - collagen:                       38 %
      - heteropolysaccharide:           1%
      - water:                          3-8 %
      - ions:                           55% (chiefly hydroxyapatite, or Ca10(PO4)6(OH)2]
- blood [Ca2+] levels
   - effective concentration depends on unbound [Ca2+]
   - maintained within narrow limits
      - plasma [Ca2+]: 10 mg/100 mL (50% of this is complexed with albumin)
      - fluctuate less than ± 3% in healthy individuals, regardless of intake and output
   - restored to normal via the actions of three hormones
      - calcitonin
      - cholecalciferol (vitamin D)
      - parathyroid hormone (PTH)
- consequences of Ca2+ imbalance
   - low [Ca2+]: tetany (irritability)
   - high [Ca2+]: respiratory, cardiac failure
- maintaining blood Ca2+ levels
   - methods
      - dissociation of plasma protein-Ca2+: equilibrium process
      - mobilization of Ca2+ from the bone: hormonal process
      - altered renal excretion of Ca2+:        hormonal process; 99% reabsorbed without PTH
                                                                       Biochemistry: NOTES & OBJECTIVES (page 161 of 165)



     - altered absorption from the intestine: hormonal process; active transport promoted by vitamin D
  - hormonal regulation
     - calcitonin: directly proportional to plasma Ca2+
     - parathyroid hormone: inversely proportional to plasma Ca 2+

calcitonin (CT, thyrocalcitonin)
- function:           reduce blood Ca2+ levels (“tone down that calcium!”)
- structure:          single-chain polypeptide (32 amino acids)
- synthesis:          produced by the C cells of the thyroid
- regulation:         solely based on serum Ca2+ levels, with increased blood Ca2+ giving increased secretion
                      - half life: 4-12 minutes
- effects:            bone: increased deposition, decreased release (targets osteoclasts)
                      kidney: increases excretion of Ca2+ and Pi, decreases reabsorption
- mechanism:          Gs-linked elevation of cAMP in target cells
- clinical:           no known associated human disease with deficiency or excess, suggesting compensatory factors
parathormone (parathyroid hormone, PTH)
- function:           increase blood Ca2+ levels (nothing cute here; simply opposes calcitonin)
- structure:          single chain polypeptide (84 amino acids)
- synthesis:          produced by the parathyroid gland; precursor is processed, stored in granules
- regulation:         solely based on serum Ca2+ levels, with decreased blood Ca2+ giving increased secretion
                      - half life: 20-30 minutes
- effects:            bone: stimulation of osteolysis, inhibition of collagen synthesis, causing Ca 2+ release
                      kidney: increases Ca2+ in distal tuble; decreases phosphate reabsorption
                      - by mass action, phosphate is drawn from hydroxyapatite in bone, releasing Ca 2+
                      - also activates 1α-hydroxylase activity, leading to production of 1,25-dihydrocholecalciferol
                      - 1,25-dihydrocholecalciferol: active D3 hormone form that promotes intestinal Ca2+ reabsorption
- mechanism:          Gs-linked elevation of cAMP in target cells (targets different cells than calcitonin)
                      Gq-linked phosphoinositide hydrolysis
- clinical:           hypoparathyroidism: rare; usually follows certain surgeries
                      hyperparathyroidism: can proceed from hyperplasia, cancer, or iatrogenic causes
cholecalciferol (Vitamin D3)
- general considerations
   - structure:       steroid-like
   - synthesis:       can be synthesized from cholesterol derivatives (and is not a vitamin in the strict sense)
                      - cholecalciferol: D3; form found in humans, derived from animal products
                      - ergocalciferol: D2; form found in plants
   - metabolism: 7-dehydrocholesterol  cholecalciferol (D3) [UV irradiation]
                      - 7-dehydrocholesterol: intermediate in cholesterol synthesis
                      - cholecalciferol: precursor to active form in humans
   - clinical:        rickets: disease caused by lack of active vitamin D
                      - synthesis from cholesterol is the primary source in an non-supplemented diet
                      - now rare, but used to be prevalent in areas with long winters and poor diet
- cholecalciferol as a vitamin
   - vitamin deficiency: two classes
      - involving the organic matrix
      - involving the mineral components (e.g. rickets, osteomalacia)
   - diseases
      - rickets
         - rickets: deficient bone calcification in children, caused by vitamin D deficiency
         - cause: improper calcification of cartilaginous structures in developing bone, leading to poor stability
         - treatment: dietary fortificaiton, increased sunlight
      - osteomalacia
         - osteomalacia: adult form of rickets
         - cause: same as rickets; more prevalent in those with poor diets, Ca2+ stores depleted by many pregnancies
   - cholecalciferol metabolism
      - requirements: 20 μg daily
                                                                        Biochemistry: NOTES & OBJECTIVES (page 162 of 165)



      - storage: liver can store a 3 week supply
      - toxicity: excessive intake can cause bone demineralization, peripheral calcification (kidney, soft tissue)
- cholecalciferol as a hormone or prohormone
   - function:       proper mineralization of bone, maintenance of adequate plasma Ca 2+
   - synthesis:      7-dehydrocholesterol  cholecalciferol (D3) [UV irradiation]
                     cholecalciferol  25-hydroxycholecalciferol [25-hydroxylase: liver]
                               - major circulating precursor
                     in presence of low Ca2+:
                     25-hydroxycholecalciferol  1α,25-dihydroxycholecalciferol [1-hydroxylase: kidney]
                               - most active form of vitamin D3
                     1α,25-DHCC  1α,24,25-trihydroxycholecalciferol [24-hydroxylase: kidney]
                               (1,24,25-THCC: less active form of vitamin D3)
                     in presence of normal or high Ca2+:
                     25-hydroxycholecalciferol  24,25-dihydroxycholecalciferol [1-hydroxylase: kidney]
                               (24,25-DHCC: inactive form of vitamin D3)
                     24,25-DHCC  1α,24,25-trihydroxycholecalciferol [24-hydroxylase: kidney]
                               (1,24,25-THCC: less active form of vitamin D3)
   - regulation: PTH, low Ca2+: enhances expression of 1α-hydroxylase (diverts to active vitamin D)
                     high Ca2+: diverts to inactive, less active forms of vitamin D
   - effects:        general steroid transcriptional model
   - targets:        intestine: stimulates Ca2+ absorption
                     bone: mobilization of Ca2+, which raises [Ca2+], stimulating resynthesis by osteoblasts
                     kidney: along with PTH, increases renal absorption of Ca 2+
                     others: possible link to immune system function
   - metabolism: not readily excreted; in low Ca2+, converted to 1,24,25-THCC, which only affects intestine

TABLE: Summary of Hormonal Control of [Ca2+] and Phosphate
 actions       calcitonin                    parathyroid hormone                         1,25-DHCC
            2+
 serum [Ca ] ↓                               ↑                                           ↑
 serum [Pi]    ↓                             ↓                                           ↑
 kidney        ↓ Ca2+ reabsorption           ↑ reabsorption of Ca2+                      ↑ Ca2+ reabsorption
               ↓ Pi reabsorption             ↓ reabsorption of Pi                        ↑ Pi reabsorption
                                             ↑ 1α-hydroxylase activity
 bone          ↓ resorption of bone          ↑ resorption of bone                        ↑ Ca2+ mobilization from bone
 GI tract      no effect                     ↑ absorption of Ca2+, Pi                    ↑ absorption of Ca2+, Pi
                                             (indirect: increased production
                                             of 1,25-DHCC)




66. Thyroxine and Triiodothyronine
Study Guide
thyroid hormones
- function:     modulate basal metabolic rate, general metabolism
- synthesis:    iodide accumulation:       TSH-stimulated active transport
                iodide oxidation:          thyroid peroxidase-catalyzed conversion from I to I+
                tyrosine iodination:       iodinase-catalyzed iodination of thyroglobulin
                tyrosine conjugation:      coupling of two iodinated tyrosine residues to form threonine
- secretion:    storage:                   maintained in follicular lumen as thyroglobulin
                proteolysis:               TSH-stimulated proteolysis, releasing T 3 and T4
                secretion
- regulation:   negative feedback: T3, T4 prevent release of TRF, TSH
                                                                      Biochemistry: NOTES & OBJECTIVES (page 163 of 165)



                   iodide (permissive): sufficient iodide required in thyroid for synthesis to occur
                   transport: thyroxine-binding globulin (TBG), thyroxine-binding prealbumin (TBP), albumin
- metabolism:      phenolic conjugation: major degradation step; uses glucouronic acid, sulfate moieties
- mechanism:       analogous to steroid receptors: directly enter cells, modulate transcriptional processes
- effects:         calorigenic effect: increased BMR and O2 consumption, with accompanying thermogenesis
                   - increase ATP utilization in energy-requiring processes, stimulate Na+/K+-ATPase
                   - increased mitochondria (size, number, cristae)
                   metabolic effects: anabolic at normal levels
                   - protein metabolism: stimulate general protein synthesis; at high levels, protein breakdown
                         - stimulate transcriptional processes, also promote GH production
                   - lipid metabolism: increases synthesis, degradation of lipids, with predomination of catabolism
                   - carbohydrate metabolism: T3 and T4 levels lead to insulin degradation, giving expected effects
                         - high T3, T4: degrade insulin, facilitate effects of adrenaline
                         - gluconeogenesis: increase available precursors from protein, fat breakdown
                         - β-adrenergic receptors: increase sensitivity (increase number, decrease Gi)
                         - decrease liver glycogen, increase muscle glycogen and glucose uptake
                         - low T3, T4: insulin predominates, giving excessive fat buildup
                   - growth and development: stimulate normal growth, within limits (catabolic in excess)
                         - increased O2 consumption
                         - increased catabolism
                         - increased negative nitrogen balance
diseases of the thyroid
- hypothyroidism
   - cretinism: severely deficient growth and mental development in children
   - myxedema: low BMR, swelling of the face and hands, obesity in adults; can be treated with thyroid hormone
   - goiter: hyperplasia of the thyroid gland due to lack of iodine; may or man not have hypothyroidism
- hyperthyroidism
   - adenomas: increased BMR, appetite, excessive nervousness, excessive sweating, heat sensitivity
   - exophthalmic goiter (Graves’ disease): autoimmune; diffuse enlargement of thyroid, hyperthyroidism


Notes: Lecture and Reading
thyroid hormones: introduction
- thyroid hormones
   - calcitonin: used in proper Ca2+ metabolism
   - thyroxine (T4), triiodothyronine (T 3): “thyroid hormones”
      - critical for normal metabolism, growth, and development
      - iodinated tyrosine derivatives, where large I atoms make the hormones quite hydrophobic
      - effects: stimulation of metabolism, maturation effects
biological effects
- overview
   - basal metabolic rate (BMR): increased by thyroid hormones
      - anabolic at normal levels
      - catabolic at high levels (limit to growth-inducing effects)
   - time of action: require hours or days to exert effects
   - imbalances
      - normal: anabolic
      - hyperthyroidism: catabolic; elevated rate of most cellular processes, including protein breakdown
      - hypothyroidism: slowed rate of most cellular processes, with low BMR and body temperature
- calorigenic effect: increased BMR and O2 consumption, with accompanying thermogenesis
   - increase ATP utilization in energy-requiring processes
      - increased mitochondria (size, number, cristae)
      - stimulate Na+/K+-ATPase
   - resulting ATP hydrolysis is responsible for the increase in O2 uptake, heat production
- metabolic effects: anabolic at normal levels
   - protein metabolism: stimulate general protein synthesis; at high levels, protein breakdown
                                                                       Biochemistry: NOTES & OBJECTIVES (page 164 of 165)



      - stimulate transcriptional processes
      - directly, indirectly promote GH production, which also stimulates transcriptional processes
   - lipid metabolism: increases synthesis and degradation of lipids, with predomination of the catabolic effect
      - hypothyroid state: decreased lipolysis
      - hyperthyroid state: increased lipolysis
         - appears similar to diabetes mellitus
         - however, also presents with fever and high BP, which diabetes usually will not
   - carbohydrate metabolism: T3 and T4 levels lead to insulin degradation, giving expected effects
      - high T3, T4: degrade insulin, facilitate effects of adrenaline
         - gluconeogenesis: increase available precursors from protein, fat breakdown
         - β-adrenergic receptors: increase sensitivity (increase number, decrease G i)
         - decrease liver glycogen, increase muscle glycogen and glucose uptake
      - low T3, T4: insulin predominates, giving excessive fat buildup
- growth and development: stimulate normal growth
   - cretinism: linked to thyroid deficiency
      - permissive effect to GH
      - will only stimulate growth to normal levels, as opposed to GH
   - excess doses: catabolic effect
      - increased O2 consumption
      - increased catabolism
      - increased negative nitrogen balance
      - weight loss (thyrotoxic effect)
      - in too much excess, can lead to cardiac arrest through β receptors
mechanism of thyroid hormone action
- active form: T3 (most peripheral tissues convert T 4 to T3)
- mechanism: analogous to steroid receptors
   - enter target cells, bind to receptors associated with nuclear chromatin
   - there are cytosolic receptors, though their purpose is not clear
thyroglobulin
- thyroglobulin: precursor storage form of iodinated thyroid hormones
   - structure: large glycoprotein (MW = 660,000)
   - hydrolysis leads to several iodine-containing derivatives of tyrosine
- derivatives
   - monoiodotyrosine          (MIT)
   - diiodotyrosine            (DIT)
   - diiodothyronine           (3,3’)
   - triiodothyronine          (3,5,3’) (T3)
   - thyroxine                 (3,5,3’,5’) (T 4)
   - triiodothyronine          (3,3’,5’) (T3, reversed)
metabolism
- synthesis
   - iodide accumulation: TSH-stimulated active transport
   - iodide oxidation:         thyroid peroxidase-catalyzed conversion from I to I+
   - tyrosine iodination: iodinase-catalyzed iodination of thyroglobulin
   - tyrosine conjugation: coupling of two iodinated tyrosine residues to form threonine
- secretion
   - storage:                  maintained in follicular lumen as thyroglobulin
   - proteolysis:              TSH-stimulated proteolysis, releasing T 3 and T4
   - secretion
- regulation
   - negative feedback:        T3, T4 prevent release of TRF, TSH
   - iodide:                   sufficient iodide required in thyroid for synthesis to occur
- transport
   - thyroxine-binding globulin (TBG):            binds T4 more tightly than T3
   - thyroxine-binding prealbumin (TBP): binds only to T4
   - albumin:                                     binds both T3 and T4 if TBG and TBPA sites are saturated
                                                                   Biochemistry: NOTES & OBJECTIVES (page 165 of 165)



- degradation
   - phenolic conjugation: major degradation step; uses glucouronic acid, sulfate moieties
   - removal of iodide: thyroid dehalogenase
   - oxidative deamination, transamination
   - half lives
      - T4: 6-7 days
      - T3: 2-3 days
diseases of the thyroid
- hypothyroidism
   - cretinism: severely deficient growth and mental development in children
   - myxedema: low BMR, swelling of the face and hands, obesity in adults; can be treated with thyroid hormone
   - goiter: hyperplasia of the thyroid gland due to lack of iodine; may or man not have hypothyroidism
- hyperthyroidism
   - adenomas: increased BMR, appetite, excessive nervousness, excessive sweating, heat sensitivity
   - exophthalmic goiter (Graves’ disease): autoimmune; diffuse enlargement of thyroid, hyperthyroidism
antithyroid agents
   - prevent hormone release:          feedback effects (thyroxine, analogs)
   - inhibit hormone synthesis:        interfere with I- uptake (SCN-, NO3-, C104)
                                       interfere with iodinaiton (iodide, thiourea, thiouracil)
                                       interfere with coupling (thiourea, thiouracil)
   - interfere with metabolism:        thyronine analogs
   - irradiation:                      damage to thyroid tissue using I131 or X-rays

				
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posted:8/7/2012
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