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Glycolysis_ gluconeogenesis and pentose phosphate pathway

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					  Glycolysis, gluconeogenesis
and pentose phosphate pathway




                           1
• Glycolysis
• Feeder pathway for glycolysis
• Fates of pyruvate under anaerobic
  conditions: fermentation
• Gluconeogenesis
• Pentose phosphate pathway


                                      2
            Glycolysis Overview
• The glycolytic pathway describes the oxidation of
  glucose to pyruvate with the generation of small
  amount of ATP and NADH

• Glycolysis is a universal pathway; present in all
  organisms: from yeast to mammals.

• In eukaryotes, glycolysis takes place in the
  cytosol



                                                      3
• Glycolysis is anaerobic; it does not require
  oxygen.

• In the presence of O2, pyruvate is further
  oxidized to CO2.

• In the absence of O2, pyruvate can be fermented
  to lactate or ethanol.



                                                 4
                           Glucose


          10 steps in
           2 phases                      3 regulated steps


                                         No O2
                           Pyruvate              Lactate


                Energy (ATP) and metabolites
Net Reaction:

Glucose + 2NAD+ + 2 Pi + 2ADP = 2 Pyruvate + 2ATP + 2 NADH + 2H2O



                                                                    5
6
7
Inputs:     Outputs:
1 Glucose   2 pyruvate
2 NAD+      2 NADH
2 ATP       2 ADP
4 ADP       2 ATP (net gain)
2P

                          8
9
• The required adjustment in the rate of glycolysis
  is achieved by a complex interplay among
  – ATP consumption,
  – NADH regeneration
  – Allosteric regulation of several glycolytic enzymes:
    hexokinase (step 1), PFK-1 (step 3), and pyruvate
    kinase (step 10)
  – Second-to-second fluctuations in the concentration of
    key metabolites that reflect the cellular balance
    between ATP production and consumption

                                                        10
Step Enzyme           Inhibitors    Stimulators
 1   Hexokinase     Epinephrine +
                      Glucagon        Insulin
                     (low blood
                      glucose)
 3     Phospho           ATP           AMP
     fructokinase        [H+]        F-2,6-BP
                        citrate

10    Pyruvate          ATP          F1,6-BP
       kinase         Glucagon

                                                  11
  Feeder pathway for glycolysis
• Many carbohydrates besides glucose meet their
  catabolic fate in glycolysis
  – Glycogen and starch enter glycolysis in a two-step
    process.
  – Ingested polysaccharides and disaccharides are
    converted to monosaccharides by intestinal hydrolytic
    enzymes
  – A variety of D-hexoses, including fructose, galactose,
    and mannose, can be phosphorylated and converted
    to either glucose 6-phosphate or fructose 6-
    phosphate
                                                         12
13
Fates of pyruvate under anaerobic
     conditions: fermentation
• Under anaerobic conditions, pyruvate can
  be used in
  – Lactic acid fermentation
  – Alcohol fermentation




                                             14
• Lactic acid fermentation occurs in a variety
  of microorganisms or in the cells of higher
  organisms when oxygen supply is limitted.




                                             15
• Yeast and other microorganisms ferment
  glucose to ethanol and CO2, rather than to
  lactate




                                           16
• In fermentation, NADH formed in glycolysis
  is used to regenerate NAD by transferring
  electrons from NADH to pyruvate, forming
  lactate or athanol

• There is no net oxidation or reduction of the
  carbons of glucose.


                                              17
         Gluconeogenesis
• Glucose is the nearly universal fuel and
  building block in modern organisms, from
  microbes to humans
• In mammals, some tissues depend almost
  completely on glucose for their metabolic
  energy.
  – For examp: The brain alone requires about
    120 g of glucose each day—more than half of
    all the glucose stored as glycogen in muscle
    and liver
                                               18
• The supply of glucose is not always sufficient;
  between meals and during longer fasts, or after
  vigorous exercise, organisms need a method for
  synthesizing glucose from noncarbohydrate
  precursors.

• This is accomplished by a pathway called
  gluconeogenesis, which converts pyruvate and
  related three- and four-carbon compounds to
  glucose.

                                                19
• Noncarbohydrate precursors of glucose
  enter the pathway chiefly at
  – pyruvate,
  – oxaloacetate
  – dihydroxyacetone phosphate




                                          20
• The major noncarbohydrate precursors are:
  – Lactate: formed by active skeletal muscles when the
    rate of glycolysis exceeds the metabolic rate of the
    citric acid cycle and the respiratory chain.
  – Amino acids: derived from proteins in the diet and
    during starvation, from the breakdown of protein in
    the skeletal muscles.
  – Glycerol: derived from the hydrolysis of
    triacylglycerols in fat cells



                                                           21
• Gluconeogenesis occurs in all animals,
  plants, fungi, and microorganisms.

• The reactions are essentially the same in
  all tissues and all species.




                                              22
• Gluconeogenesis is not a reversal of
  glycolysis:
  – Glycolysis: glucose to pyruvate;
    gluconeogenesis: pyruvate to glucose
  – Gluconeogenesis shares 7 steps with
    glycolysis, but has bypasses 3 steps




                                           23
Pyruvate kinase



                  24
      Hexokinase




Phosphofructokinase




                      25
Enzymatic differences between glycolysis
and glyconeogenesis

     Glycolysis            Gluconeogenesis
Hexokinase            Glucose 6-phosphatase

Phosphofructokinase   Fructose 1,6-bisphosphatase

Pyruvate kinase       Pyruvate carboxylase
                      Phosphoenolpyruvate
                      carboxykinase
                                               26
1. Phosphoenolpyruvate is formed from
   pyruvate by way of oxaloacetate in two
   steps in mitochondria:
  – Pyruvate is carboxylated to oxaloacetate at
    the expense of an ATP
  – Oxaloacetate is decarboxylated and
    phosphorylated to yield phosphoenolpyruvate
    at the expense of a GTP


                                             27
                         pyruvate
                         carboxylase
pyruvate + CO2+ ATP + H2O         oxaloacetate + ADP + Pi + 2 H+
                phosphoenolpyruvate
                carboxykinase
oxaloacetate + GTP          phosphoenolpyruvate + GDP + CO2

pyruvate + ATP + GTP + H2O      oxaloacetate + ADP + GDP + Pi + 2 H+




                                                              2-


                                                    -

                                          Phosphoenolpyruvate
                                                                   28
2. Fructose 6-phosphate is formed from
   fructose 1,6-bisphosphate by hydrolysis
   the phosphate ester at C-1
                             fructose 1,6-
                             bisphosphatase
fructose 1,6-bisphosphate + H2O       fructose 6-phosphate + Pi




   fructose 6-phosphate           fructose 1,6-bisphosphate       29
3. Glucose is formed by hydrolysis of
   glucose 6-phosphate in the lumen of
   the endoplasmic reticulum
                           glucose 6-phosphatase
         glucose 6-phosphate + H2O       glucose + Pi




                                            glucose     30
Alternative paths from pyruvate to
phosphoenolpyruvate:

      -Puruvate carboxylase is the
      mitochondrial enzyme, others
      enzymes of the gluconeogenesis
      are cytoplasmic.
      - Oxaloacetate is transported out
      of mitochondria in the form of
      malate.


      Oxaloacetate, derived from
      lactate, produced by glycolysis,
      for example in muscles, is
      converted directly to PEP in the
      liver


                                         31
         The Cori cycle




Significance:
- Prevention of lactic acidosis
- Production of ATP for the muscle by glycolysis


                                                   32
It would be the consumption of ATP (4 ATP) and the production
of heat if glycolysis and gluconeogenesis were allowed to
proceed simultaneously at high rates  both processes are
reciprocally regulated so that one pathway is relatively inactive
while the other is highly active                                33
• Local control by adenine nucleotide:
  – Phosphofructokinase (glycolysis) is inhibited by ATP
    and stimulated by AMP
  – Fructose 1,6-bisphosphatase (gluconeogenesis) is
    inhibited by AMP

   Insures that:
     - When cellular ATP is high (low AMP), glucose is not
       degraded to make ATP.
     - When cellular AMP is high (low ATP), the cells does not
       expend energy to synthesizingglucose

                                                                 34
  Pentose phosphate pathway
   (PPP) of glucose oxidation

 Also known as:
 - Pentose shunt
 - Hexose monophosphate pathway
 - Phosphogluconate pathway


Glucose 6-phosphate + 2 NADP+ + H2O
     ribose 5-phosphate + 2 NADPH + 2 H+ + CO2
                                                 35
• The pentose phosphate pathway is
  primarily an anabolic pathway that utilizes
  the 6 carbons of glucose to generate 5
  carbon sugars and reducing equivalents.

• It does oxidize glucose and under certain
  conditions can completely oxidize glucose
  to CO2 and water.
                                                36
The primary functions of this pathway are:

1. Generate NADPH (for reductive biosynthesis) –
   needed for synthesis of fatty acids and steroids:
  – Similar to NAD+, NADP+ has an extra phosphate
  – The pentose phosphate pathway generates almost all
    cellular NADPH used in reductive biosynthesis.
  – In mammals, this pathway is prominent in tissues
    actively carrying out biosynthesis of fatty acids and
    steroids from small precursor molecules – need
    NADPH
     •   Especially prevalent in the mammary gland, adipose tissue,
         the adrenal cortex and the liver.
     •   Other tissues (e.g., skeletal muscle, brain) have virtually no
         pentose phosphate activity.
                                                                     37
NAD+ vs. NADP+
- NADH is oxidized by the respiratory
chain to regenerate ATP.
- NADPH serves as an electron donor
in reductive biosynthesis.
                                 38
2. Generate Ribose 5-phosphate (R5P) for the
   biosynthesis of nucleotides, nucleic acids and
   several enzyme cofactors
- Enzymes of the pathway are located in the cytosol.
- The pathway is divided into an oxidative and
   nonoxidative branch.
   The oxidative branch produces NADPH and ribulose
    5-phosphate as G6P is oxidized.
   In the nonoxidative branch, depending on cellular
    conditions, the pentose phosphates are either converted
    to ribose-5-phosphate or converted to the glycolytic
    intermediates -fructose-6-phosphate (F6P) and
    glyceraldehyde 3-phosphate (GAP).                   39
Ribose 5-phosphate
                     40
3. Convert pentoses to hexoses, feeding into
   glycolysis. Equally important reactions of
   the PPP are to convert dietary 5 carbon
   sugars into both 6 (fructose-6-phosphate)
   and 3 (glyceraldehyde -3- phosphate)
   carbon sugars which can then be utilized by
   the pathways of glycolysis.


                                           41
42
• Oxidative phase:
  – Produces pentose phosphate and NADPH
  – Occurs in 4 steps




                                           43
Step 1: Oxidation of glucose 6-phosphate

                                   - Oxidation-Reduction
                                   Reaction
                                   - Uses NADP+ as the
                                   cofactor
                                   - Forms 6-phospho-D-
                                   gluconolactone
                                        - Lactones are cyclic
                                        esters formed when an
                                        acid and an alcohol
                                        group on the same
                                        molecule react and
                                        usually require that a 5
                                        or 6-membered ring
                                        be formed
                                   - This is the regulated step:
                                   depends on the conc. of
                                   NADPH, which is a strong
                                   inhibitor of the enzyme
                                   (product inhibition).    44
Step 2: Hydrolysis of 6-phosphoglucono δ-lactone




                                                   45
Step 3: Oxidation and decarboxylation of 6-P-gluconate




- The oxidation and decarboxylation step releases carboxyl group as CO2,
again transferring the electrons to NADP+
- 2 moles NADPH/mole G6P are formed in this portion of the pathway
                                                                       46
Step 4: Isomerization/Rearrangement by
phosphopentose isomerase




                                         47
• Nonoxidative phase
   – Cells need NADPH more than ribose 6-P
   – Ribose 6-P is converted into glyceraldehyde 3-P and fructose 6-
     P by transketolase and transaldolase
   – Net results of the reactions is the formation of two hexoses and
     one triose

                            Transketolase
             C5 + C5                         C3 + C7
                           Transaldolase
             C3 + C7                          C4 + C6
                           Transketolase
             C4 + C5                          C3 + C6

 Overall:        3 C5                         2C6 + C3
                                                                    48
Step 1: Formation of glyceraldehyde 3-P and sedoheptulose 7-P
from two pentoses




                                                                49
Step 2: Formation of erythrose 4-P and fructose 6-P from
sedoheptulose 7-P and glyceraldehyde 3-P




                                                           50
Step 3: Formation of glyceraldehyde 3-P and fructose 6-P
from xylulose 5-P and erythrose 4-P




                                                           51
• Regulation of Pentose phosphate pathway
  – Flux through PPP and rate of NADPH production is
    controlled by the rate of glucose 6-P dehydrogenase
    (G6PDH)
  – Regulated by NADP+ concentration – high concentration
    increases activates G6PDH
  – Ribulose 5-phosphate is converted to either ribose-5-
    phosphate (R5P) and/or fructose 6- phosphate (F6P)
    and glyceraldehyde 3-phosphate (GAP).
     • Both F6P and GAP can enter glycolysis or gluconeogenesis.


                                                              52
53
• The flow of glucose 6-P depends on the
  need for NADPH, ribose 5-P and ATP
  – There are four posibilities




                                           54
• More ribose-5-phosphate than NADPH needed
  – Synthesize R5P without making NADPH by by-passing the oxidative
    steps
  – Use F6P and GAP from glycolytic pathway
                                                                55
Both ribose-5-phosphate and NADPH needed
- First four reactions (oxidative phase) predominate (glucose 6-phosphate
to ribose 5-phosphate)
- All the ribulose 5-phosphate is isomerized to ribose 5-phosphate, and
the pathway is completed.
                                                                     56
More NADPH than ribose-5-phosphate is needed
- Ribose 5-P made into fructose 6-P and glyceraldehyde 3-P for entry
into gluconeogenesis
- In the case of recycling through gluconeogenesis, 1 molecule of
glucose 6-P can be converted by 6 cycles of the pentose phosphate
pathway and gluconeogenes is to 6 CO2 and 12 NADPH molecules. 57
Need NADPH and ATP, but not ribose-5-phosphate
- Ribulose 5-P converted to fructose 6-P and glyceraldehyde 3-P (in nonoxidative
phase) for glycolysis to make ATP and NADH                                     58

				
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