Enzyme Mechanisms by 9H95h7

VIEWS: 7 PAGES: 50

									     Transport & Signaling;
     Nucleic Acid Chemistry

                       Andy Howard
                 Introductory Biochemistry
                    30 September 2008

Biochemistry:Transport; Nucleic Acids        09/30/08
        What we’ll discuss
   Membrane transport            Membrane Signaling
       Energetics                    Adenylyl cyclase
       Active transport              Inositol-phospholipid
       Transporting big               signaling pathway
        molecules                     Receptor tyr kinases
                                  Nucleic acid chemistry
                                      Pyrimidines: C, U, T
                                      Purines: A, G
                                  Nucleosides


09/30/08 Biochemistry:Transport; Nucleic Acids      p. 2 of 50
     Protein-facilitated
     passive transport
   All involve negative DGtransport
        Uniport: 1 solute across
        Symport: 2 solutes, same direction
        Antiport: 2 solutes, opposite directions   Diagram courtesy
   Proteins that facilitate this are like          Saint-Boniface U.
    enzymes in that they speed up
    reactions that would take place slowly
    anyhow
   These proteins can be inhibited,
    reversibly or irreversibly


09/30/08 Biochemistry:Transport; Nucleic Acids           p. 3 of 50
 Kinetics of passive transport
    Michaelis-Menten saturation kinetics:
     v0 = Vmax[S]out/(Ktr + [S]out) …
     we’ll revisit this after we do enzyme
     kinetics
    Vmax is velocity achieved with fully
     saturated transporter
    Ktr is analogous to Michaelis constant:
     it’s the [S]out value for which half-maximal
     velocity is achieved.

09/30/08 Biochemistry:Transport; Nucleic Acids   p. 4 of 50
    Primary active
                                                QuickTime™ an d a

    transport                          TIFF (Uncompressed) decompressor
                                          are need ed to see this p icture .

   Energy source:
    usually ATP or light
   Energy source directly                  Bacteriorhodopsin
    contributes to overcoming               PDB 1F50, 1.7Å
    concentration gradient                  25 kDa monomer
       Bacteriorhodopsin: light energy used to drive
        protons against concentration and charge
        gradient to enable ATP production
       P-glycoprotein: ATP-driven active transport of
        many nasties out of the cell

09/30/08 Biochemistry:Transport; Nucleic Acids            p. 5 of 50
      Secondary active
                                                      QuickTime™ an d a
      transport                              TIFF (Uncompressed) decompressor
                                                are need ed to see this p icture .


   Active transport of one solute is
    coupled to passive transport of
    another
                                                Pyrococcus Multi-
   Net energetics is (just barely)             sugar transporter
    favorable                                   PDB 1VCI
   Generally involves antiport                 83 kDa dimer
       Bacterial lactose influx driven by
        proton efflux
       Sodium gradient often used in
        animals


09/30/08 Biochemistry:Transport; Nucleic Acids                  p. 6 of 50
    Complex case:
    Na+/K+ pump
   Typically [Kin] = 140mM, [Kout] = 5mM,
    [Nain] = 10 mM, [Naout] = 145mM.
   ATP-driven transporter:
    3 Na+ out for 2 K+ in                        Diagram courtesy
    per molecule of ATP hydrolyzed               Steve Cook
   3Na out: 3*6.9 kJmol-1,
    2K in: 2*8.6 kJmol-1
    = 37.9 kJ mol-1 needed, ~ one ATP


09/30/08 Biochemistry:Transport; Nucleic Acids      p. 7 of 50
    What’s this
    used for?
   Sodium gets pumped back
    in in symport with glucose,
    driving uphill glucose
    transport                                    Diagram courtesy
   That’s a separate passive                    Steve Cook
    transport protein called
    GluT1 to move glucose
    back

09/30/08 Biochemistry:Transport; Nucleic Acids        p. 8 of 50
 How do we transport big
 molecules?
    Proteins and other big molecules often
     internalized or secreted by endocytosis
     or exocytosis
    Special types of lipid vesicles created for
     transport



09/30/08 Biochemistry:Transport; Nucleic Acids   p. 9 of 50
     Receptor-mediated
     endocytosis
   Bind macromolecule to specific receptor in
    plasma membrane
   Membrane invaginates, forming a vesicle
    surrounding the bound molecules (still on the
    outside)
   Vesicle fuses with endosome and a lysozome
   Inside the lysozyome, the foreign material
    and the receptor get degraded
   … or ligand or receptor or both get recycled

09/30/08 Biochemistry:Transport; Nucleic Acids   p. 10 of 50
 Example: LDL-cholesterol




                                        Diagram courtesy
                                        Gwen Childs,
                                        U.Arkansas for Medical
                                        Sciences


09/30/08 Biochemistry:Transport; Nucleic Acids     p. 11 of 50
    Exocytosis


   Materials to be secreted are             Diagram courtesy
                                             LinkPublishing.com
    enclosed in vesicles by the
    Golgi apparatus
   Vesicles fuse with plasma
    membrane
   Contents released into
    extracellular space

09/30/08 Biochemistry:Transport; Nucleic Acids    p. 12 of 50
 Transducing signals
   Plasma membranes contain receptors
    that allow the cell to respond to chemical
    stimuli that can’t cross the membrane
   Bacteria can detect chemicals:
    if something useful comes along,
    a signal is passed from the receptor to
    the flagella, enabling the bacterium to
    swim toward the source


09/30/08 Biochemistry:Transport; Nucleic Acids   p. 13 of 50
 Multicellular
 signaling
    Hormones,
     neurotransmitters, growth                    Diagram
                                                  courtesy
     factors all can travel to                    Science
                                                  Creative
     target cells and produce                     Quarterly, U.
     receptor signals                             British
                                                  Columbia




09/30/08 Biochemistry:Transport; Nucleic Acids   p. 14 of 50
    Extracellular
    Signals
   Internal behavior of
    cells modulated by external influences              Image
   Extracellular signals are called first              courtesy
                                                        CSU
    messengers                                          Channel
   7-helical transmembrane proteins with               Islands
    characteristic receptor sites on
    extracellular side are common, but
    they’re not the only receptors
    09/30/08 Biochemistry:Transport; Nucleic Acids   p. 15 of 50
 Internal results of signals
    Intracellular: heterotrimeric G-proteins
     are the transducers: they receive signal
     from receptor, hydrolyze GTP, and emit
     small molecules called second
     messengers
    Second messengers diffuse to target
     organelle or portion of cytoplasm
    Many signals, many receptors,
     relatively few second messengers
    Often there is amplification involved
09/30/08 Biochemistry:Transport; Nucleic Acids   p. 16 of 50
 Roles of these systems
    Response to sensory stimuli
    Response to hormones
    Response to growth factors
    Response to some neurotransmitters
    Metabolite transport
    Immune response
    This stuff gets complicated, because the
     kinds of signals are so varied!

09/30/08 Biochemistry:Transport; Nucleic Acids   p. 17 of 50
 G proteins
     Transducers of external signals into the inside
      of the cell
     These are GTPases (GTP  GDP + Pi)
     GTP-bound protein transduces signals
      GDP-bound protein doesn’t
     Heterotrimeric proteins; association of b and g
      subunits with a subunit is disrupted by
      complexation with hormone-receptor complex,
      allowing departure of GDP & binding of GTP


09/30/08 Biochemistry:Transport; Nucleic Acids   p. 18 of 50
                                                      GTP

        G protein                  Inactive    GDP

                                      a
        cycle                                              Active
                             b
   Ternary complex            g                            a
                                                           GTP
    disrupted by binding of
    receptor complex             b                                       H2O
                                   g
   Ga-GTP interacts with
    effector enzyme                                                 Pi
   GTP slowly hydrolyzed
    away
                                                       a
   Then Ga-GDP                                        GDP
    reassociates with b,g                            Inactive

   See fig. 9.39 for details
    09/30/08 Biochemistry:Transport; Nucleic Acids      p. 19 of 50
    Adenylyl cyclase

   cAMP and cGMP:                     Cyclic
    second messengers                  AMP
   Adenylyl cyclase converts ATP to cAMP
       Integral membrane enzyme; active site faces
        cytosol
       cAMP diffuses from membrane surface through
        cytosol, activates protein kinase A
       PKA phosphorylates ser,thr in target enzymes;
        action is reversed by specific phosphatases


09/30/08 Biochemistry:Transport; Nucleic Acids   p. 20 of 50
                                                      O


                                                                    N
                                                 N



     Modulators                              O        N
                                                                    N




     of cAMP                                         caffeine       O

                                                                        H
                                                                        N
                                                                N


    Caffeine, theophylline inhibit cAMP        O      N
                                                                        N

     phosphodiesterase, prolonging cAMP’s
     stimulatory effects on protein kinase A       theophylline


    Hormones that bind to stimulatory receptors
     activate adenylyl cyclase, raising cAMP levels
    Hormones that bind to inhibitory receptors inhibit
     adenylyl cyclase activity via receptor interaction
     with the transducer Gi.

09/30/08 Biochemistry:Transport; Nucleic Acids            p. 21 of 50
                                              O


                                                      O        R2
                                         R1       O

Inositol-Phospholipid                                      O


Signaling Pathway                                          O
    2 Second messengers derived from O                    P   O-    OH
     phosphatidylinositol 4,5-bisphosphate                 O
                                                                               OH
     (PIP2)
    Ligand binds to specific receptor;                   HO
                                                                               O
     signal transduced through G protein                            HO    -O
                                                                               P    O
     called Gq
                                                                               O

    Active form activates
     phosphoinositide-specific
     phospholipase C bound to cytoplasmic
     face of plasma membrane
09/30/08 Biochemistry:Transport; Nucleic Acids            p. 22 of 50
                                                           O
                                          -O                          O-
                                                                P

     PIP2                             O   P      O-    O
                                                                     O-
                                          O                               OH
     chemistry                                                             -O
                                                                                            O-

   Phospholipase C                       HO                              O
                                                                                    P


    hydrolyzes PIP2 to inositol                                                         O
                                                       OH
    1,4,5-trisphosphate (IP3)
                                                               IP3
    and diacylglycerol                           O

   Both of these products are                                 O               R2
                                           R1
    second messengers that                             O


    transmit the signal into the                                     O


    cell
                                                                     OH


                                                      diacylglycerol


09/30/08 Biochemistry:Transport; Nucleic Acids             p. 23 of 50
                                                            O
                                             -O                      O-
                                                                P
                                         O   P    O-    O
                                                                    O-


  IP3 and calcium                            O                           OH
                                                                          -O
                                                                                       O-
                                                                               P
                                             HO                          O
                                                                                   O
                                                        OH


    IP3 diffuses through cytosol and binds to a IP3
     calcium channel in the membrane of the
     endoplasmic reticulum
    The calcium channel opens, releasing Ca2+ from
     lumen of ER into cytosol
    Ca2+ is a short-lived 2nd messenger too: it
     activates Ca2+-dependent protein kinases that
     catalyze phosphorylation of certain proteins


09/30/08 Biochemistry:Transport; Nucleic Acids         p. 24 of 50
                                                      O


                                                               O        R2
                                                 R1        O


                                                                   O



    Diacylglycerol and                                             OH


    protein kinase C                                      diacylglycerol


   Diacylglycerol stays @ plasma membrane
   Protein kinase C (which exists in
    equilibrium between soluble & peripheral-
    membrane form) moves to inner face of
    membrane; it binds transiently and is
    activated by diacylglycerol and Ca2+
   Protein kinase C catalyzes
    phosphorylation of a bunch of proteins
09/30/08 Biochemistry:Transport; Nucleic Acids            p. 25 of 50
     Control of inositol-
     phospholipid pathway
    After GTP hydrolysis, Gq is inactive so I
     no longer stimulates Phospholipase C
    Activities of 2nd messengers are
     transient
        IP3 rapidly hydrolyzed to other things
        Diacylglycerol is phosphorylated to form
         phosphatidate

09/30/08 Biochemistry:Transport; Nucleic Acids   p. 26 of 50
 Sphingolipids give rise to 2nd
 messengers
    Some signals activate hydrolases that convert
     sphingomyelin to sphingosine, sphingosine-1-P,
     and ceramide
    Sphingosine inhibits PKC
    Ceramides activates a protein kinase and a
     protein phosphatase
    Sphingosine-1-P can activate Phospholipase D,
     which catalyzes hydrolysis of
     phosphatidylcholine; products are 2nd
     messengers
09/30/08 Biochemistry:Transport; Nucleic Acids   p. 27 of 50
                                                ligands


                                                              exterior
      Receptor                                 Tyr kinase
      tyrosine kinases                         monomers        interior
   Most growth factors
    function via a pathway
    that involves these
    enzymes
   In absence of ligand, 2
    nearby tyr kinase
    molecules are separated
   Upon substrate binding
    they come together, form
    a dimer
    09/30/08 Biochemistry:Transport; Nucleic Acids     p. 28 of 50
Autophosphorylation                      P               P
of the dimer
    Enzyme catalyzes phosphorylation of
     specific tyr residues in the kinase itself;
     so this is autophosphorylation
    Once it’s phosphorylated, it’s activate
     and can phosphorylate various cytosolic
     proteins, starting a cascade of events


09/30/08 Biochemistry:Transport; Nucleic Acids   p. 29 of 50
    Insulin receptor
   Insulin binds to an a2b2
    tetramer;
    binding brings b subunits
    together
   Each tyr kinase (b) subunit
    phosphorylates the other one
   The activated tetramer can
    phosphorylate cytosolic
    proteins involved in metabolite              Sketch courtesy of
    regulation                                   Davidson College,
                                                 NC

09/30/08 Biochemistry:Transport; Nucleic Acids       p. 30 of 50
                                                        6
                                                 5                1
                                                              N


  Pyrimidines                                    4                2
                                                        N
                                                        3
     Single-ring nucleic acid bases           pyrimidine
     6-atom ring; always two nitrogens in the ring,
      meta to one another
     Based on pyrimidine, although pyrimidine itself
      is not a biologically important molecule
     Variations depend on oxygens and nitrogens
      attached to ring carbons
     Tautomerization possible
     Note line of symmetry in pyrimidine structure


09/30/08 Biochemistry:Transport; Nucleic Acids       p. 31 of 50
                                                        H
                                         O              N            O



    Uracil and thymine                        HN

   Uracil is a simple dioxo
                                                      uracil
    derivative of pyrimidine:
    2,4-dioxopyrimidine
                                                 HN
   Thymine is 5-methyluracil
   Uracil is found in RNA;               O             N            O
    Thymine is found in DNA                             H

   We can draw other                                 thymine

    tautomers where we move
    the protons to the oxygens
09/30/08 Biochemistry:Transport; Nucleic Acids         p. 32 of 50
                                     H
                          O          N           O                   NH
     Tautomers
                              HN                 O             N            O
   Lactam and                                           uracil - lactim
    Lactim forms               uracil - lactam   H


   Getting these right
    was essential to          HN
    Watson & Crick’s                                     HN

    development of        O          N           O
                                     H
    the DNA double                                   O          N           OH
                              thymine - lactam
    helical model                                        thymine - lactim



09/30/08 Biochemistry:Transport; Nucleic Acids                p. 33 of 50
                                                   H
                                        O          N        NH2



 Cytosine                                   N


                                                 cytosine

    This is 2-oxo,4-aminopyrimidine
    It’s the other pyrimidine base found in
     DNA & RNA
    Spontaneous deamination (CU)
     we’ll see the significance of that later
    Again, other tautomers can be drawn

09/30/08 Biochemistry:Transport; Nucleic Acids         p. 34 of 50
 Cytosine:
 amino and imino forms
   Again, this tautomerization needs to be
    kept in mind
                H
    O           N         NH2                 H
                                  O           N         NH


           N
                                         N

        cytosine -amino form          cytosine -imino form


09/30/08 Biochemistry:Transport; Nucleic Acids        p. 35 of 50
                                                 6           7
                                                             H
                                                         5   N
                                          1N
                                                                 8
                                                     4
                                          2
 Purines                                         N
                                                             N
                                                             9
                                                 3
     Derivatives of purine; again, the purine
      root molecule isn’t biologically
      important
     Six-membered ring looks a lot like
      pyrimidine
     Numbering works somewhat
      differently: note that the glycosidic
      bonds will be to N9, whereas it’s to
      N1 in pyrimidines

09/30/08 Biochemistry:Transport; Nucleic Acids       p. 36 of 50
     Adenine
    This is 6-aminopurine
    Found in RNA and DNA
    We’ve seen how important adenosine
     and its derivatives are in metabolism
    Tautomerization happens here too
      NH2                         NH

                H                            H
                N                            N
 N                          HN



                N                            N
      N                           N

 adenine - amino form        adenine - imino form
09/30/08 Biochemistry:Transport; Nucleic Acids      p. 37 of 50
    Guanine
 This is 2-amino-6-oxopurine
 Found in RNA, DNA
 Lactam, lactim forms
             O            OH

                          H                          H
                          N                          N
       HN                             N


                          N                          N
 H2N         N                 H2N         N
       guanine - lactam            guanine - lactim
09/30/08 Biochemistry:Transport; Nucleic Acids      p. 38 of 50
Other natural purines
   Hypoxanthine and xanthine
    are biosynthetic precursors
    of A & G
   Urate is important in
    nitrogen excretion
    pathways




09/30/08 Biochemistry:Transport; Nucleic Acids   p. 39 of 50
 Tautomerization and H-bonds
   Lactam forms predominate at neutral pH
   This influences which bases are H-bond
    donors or acceptors
   Amino groups in C, A, G make H-bonds
   So do ring nitrogens at 3 in pyrimidines
    and 1 in purines
   … and oxygens at 4 in U,T, 2 in C, 6 in G


09/30/08 Biochemistry:Transport; Nucleic Acids   p. 40 of 50
                                              O         NR1R2



                               HO
                                                       OH
 Nucleosides                             HO

                                 N-glycoside of ribofuranose

    As mentioned in ch. 8, these are
     glycosides of the nucleic acid bases
    Sugar is always ribose or deoxyribose
    Connected nitrogen is:
        N1 for pyrimidines (on 6-membered ring)
        N9 for purines (on 5-membered ring)


09/30/08 Biochemistry:Transport; Nucleic Acids       p. 41 of 50
        Pyrimidine nucleosides
     Drawn here in amino and lactam forms
                            OH                              OH
             HO                           HO
                                 OH                                OH



             N          O                 N             O



H2N      N        O              O    N         O
                                      H
             cytidine
                                              uridine




09/30/08 Biochemistry:Transport; Nucleic Acids               p. 42 of 50
                                 Pyrimidine
             H
                            OH   deoxynucleosides                                   OH
                                                                       H
                                       OH
                                                                                          OH



           N           O
                                                                      N         O



O     N           O                                   O        N           O
      H                                          OH
                                                               H
          2'-deoxyuridine                                          2'-deoxythymidine
                                                          OH



                                   N         O



                 H2N        N          O

                                 deoxycytidine

    09/30/08 Biochemistry:Transport; Nucleic Acids                                  p. 43 of 50
 A tricky nomenclature issue
   Remember that thymidine and its
    phosphorylated derivatives ordinarily
    occur associated with deoxyribose, not
    ribose
   Therefore many people leave off the
    deoxy- prefix in names of thymidine and
    its derivatives: it’s usually assumed.


09/30/08 Biochemistry:Transport; Nucleic Acids   p. 44 of 50
      Purine nucleosides
     Drawn in amino and lactam forms
      NH2
                                               O


                 N                                       N
 N                                        HN


                 N                                       N
      N                             H2N        N


                           O                                    O
          HO                                       HO

                                                                         OH
                               OH

                                                        HO
               HO
                                                    guanosine
               adenosine

09/30/08 Biochemistry:Transport; Nucleic Acids                      p. 45 of 50
 Purine deoxynucleosides
        NH2                                     O



                  N                                         N
                                           HN
    N


                                                            N
                  N                 H 2N        N
        N

                                                                     O
                         O

                                                                         OH
                               OH

                                                          HO
                 HO
                                                    deoxyguanosine
              deoxyadenosine


09/30/08 Biochemistry:Transport; Nucleic Acids                  p. 46 of 50
 Conformations around the
 glycosidic bond
    Rotation of the base around the glycosidic bond
     is sterically hindered
    In the syn conformation there would be some
     interference between the base and the 2’-
     hydroxyl of the sugar
    Therefore pyrimidines are always anti, and
     purines are usually anti
    Furanose and base rings are roughly
     perpendicular

09/30/08 Biochemistry:Transport; Nucleic Acids   p. 47 of 50
 Glycosidic bonds

    This illustrates the
     roughly perpendicular
     positionings of the
     base and sugar rings




09/30/08 Biochemistry:Transport; Nucleic Acids   p. 48 of 50
 Solubility of nucleosides and
 lability of glycosidic linkages
    The sugar makes nucleosides more
     soluble than the free bases
    Nucleosides are generally stable to basic
     hydrolysis
    Acid hydrolysis:
        Purines: glycosidic bond fairly readily
         hydrolized
        Pyrimidines: resistant to acid hydrolysis
09/30/08 Biochemistry:Transport; Nucleic Acids   p. 49 of 50
 Chirality in nucleic acids
    Bases themselves are achiral
    Four asymmetric centers in
     ribofuranose, counting the glycosidic
     bond.
    Three in deoxyribofuranose
    Glycosidic bond is one of those 4 or 3.
    Same for nucleotides:
     phosphates don’t add asymmetries

09/30/08 Biochemistry:Transport; Nucleic Acids   p. 50 of 50

								
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