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					                   Glycolysis
• Overall:
   conversion:       glucose ® 2 pyruvate
   with generation of ATP (2, in this case)
• These reactions are the way in which carbohydrate is
  broken down into smaller units.
• Anaerobic metabolism: The pyruvate is converted to
  lactate or ethanol (to regenerate NAD+). This is often
  referred to as fermentation.
• Aerobic metabolism: the pyruvate is fed into the TCA
  (tricarboxylic acid) cycle, where it is fully oxidized to
  CO2, releasing far more energy (~ 25 ATP/glucose).
                  Pools
  The flow of carbon through the
  glycolytic pathway may be thought of as
  the flow from one pool of intermediates
  to another. Within each pool the
  molecules may be considered to be in
  equilibrium.
1)         Hexose monophosphates:
    glucose-1-phosphate (Glc-1-Pi)
    glucose-6-phosphate (Glc-6-Pi)
    fructose-6-phosphate (Frc-6-Pi)
                      Pools
     The flow of carbon through the
     glycolytic pathway may be thought of as
     the flow from one pool of intermediates
     to another. Within each pool the
     molecules may be considered to be in
     equilibrium.
2)       Fructose bisphosphate & triose
     phosphates:
       fructose-1,6-bisphosphate (Frc-1,6-bisPi)
       glyceraldehyde-3-phosphate (G3P)
       dihydroxyacetone phosphate (DHAP)
                 Pools
  The flow of carbon through the
  glycolytic pathway may be thought of as
  the flow from one pool of intermediates
  to another. Within each pool the
  molecules may be considered to be in
  equilibrium.
3)         Phosphoglycerates & PEP:
    glycerate-3-phosphate
    glycerate-2-phosphate
    phosphoenolpyruvate (PEP)
              Pool 1: Hexose
             monophosphates
•       Entry:
    •    Mobilization of storage carbohydrate by
         glycogen phosphorylase
         (phosphorolysis of glycosidic bond
         to Glc-1-Pi)

    •    Phosphorylation of glucose by
         hexokinase
         (uses ATP to make Glc-6-Pi)
              Pool 1: Hexose
             monophosphates
•       Pool enzymes:
    •    Phosphoglucomutase: Glc-1-Pi D Glc-6-Pi
         Enzyme begins phosphorylated, and there is
         a transient intermediate where the
         dephosphorylated enzyme is bound to G-1,6-Pi.
               Pool 1: Hexose
              monophosphates
•       Pool enzymes:
    •    Phosphoglucomutase: Glc-1-Pi D Glc-6-
         Pi
         Enzyme begins phosphorylated, and there
         is
         a transient intermediate where the
         dephosphorylated enzyme is bound to G-
         1,6-Pi.

    •    Phosphohexoisomerase: Glc-6-Pi D Frc-
         6-Pi
         Could be called "Hexose phosphate
                     Pool 2:
Fructose bisphosphate & triose phosphates

  •   Entry:
      Phosphofructokinase:
        Frc-6-Pi + ATP ® Frc-1,6-bisPi + ADP
        Phosphorylates Frc-6-Pi to Frc-1,6-bisPi
        using ATP as phosphate donor.
                       Pool 2:
Fructose bisphosphate & triose phosphates
  •       Pool enzymes:
      •    Aldolase: Frc-1,6-bisPi D G3P + DHAP
           Catalyzes reverse of aldol condensation.
           (Specific for substituents at C-1 to C-4.)

      •    Triose phosphate isomerase:
           G3P D DHAP
           Allows conversion of DHAP into G3P,
           which will be used in further reactions
                       Pool 2:
Fructose bisphosphate & triose phosphates
  •       Pool enzymes:
      •    Aldolase: Frc-1,6-bisPi D G3P + DHAP
           Catalyzes reverse of aldol condensation.
           (Specific for substituents at C-1 to C-4.)

      •    Triose phosphate isomerase:
           G3P D DHAP
           Allows conversion of DHAP into G3P,
           which will be used in further reactions
    Pool 3: Phosphoglycerates
•       Entry:
    •    Phosphoglyceraldehyde
         dehydrogenase:
         G3P + NAD+ + Pi ®
                  glycerate-1,3-bisPi + NADH

         Enzyme cysteine forms thiohemiacetal
         with G3P, which is oxidized to a thioester
         by NAD+. The thioester is phosphorylzed,
         conserving some of the energy in the acid
         anhydride.
    Pool 3: Phosphoglycerates
•   Entry:

    2. 3-Phosphoglycerate kinase:
       glycerate-1,3-bisPi + ADP
                        ® glycerate-3-Pi+ ATP

       The energy in the acid anhydride is used
       to phosphorylate ATP. This pays back the
       debt incurred in making Frc-1,6-bisPi,
       although those phosphates are still
       esterified to C-1.
    Pool 3: Phosphoglycerates
•       Pool enzymes:
    •    Phosphoglyceromutase:
         glycerate-3-Pi D glycerate-2-Pi
         Mechanism similar to that of
         phosphoglucomutase.
     Pool 3: Phosphoglycerates
•   Pool enzymes:
    1. Phosphoglyceromutase:
       glycerate-3-Pi D glycerate-2-Pi
       Mechanism similar to that of
       phosphoglucomutase.

    2. Enolase:
       glycerate-2-Pi D PEP + H2O
       Dehydrates glycerate-2-phosphate, making
       high-energy PEP. The enolate form of
       pyruvate is trapped by the phosphoester;
       hydrolysis of this phosphoester is very
       favorable.
    Pool 3: Phosphoglycerates
•   Exit:

    Pyruvate kinase:
    PEP + ADP ® pyruvate + ATP

    Glycolysis finally turns a profit!
    The phosphates originally esterified to
    hexoses are now used to make ATP.
      Free energy changes
• The PFK (phosphofructokinase) and PK
  (pyruvate kinase) reactions are
  irreversible. They drive the entire
  pathway.
• The actual free energy changes (DG) are
  near zero for the all the reactions in
  between.
• Note the effect of the large cellular
  ATP/ADP ratio on all the reactions
  involving ATP.
            The Aftermath
•   Once you have made 2 pyruvates
    from each starting glucose, you have
    also:
    – phosphorylated 2 ADP ® 2 ATP
    – reduced 2 NAD+ ® 2 NADH


•   In order for the production of ATP to
    continue, it is necessary to reoxidize
    NADH, or the cell will run out of NAD+.
    (And the G3P DH reaction will stop.)
      Reoxidation of NADH
There are 3 ways to deal with pyruvate:
1)        Pyruvate dehydrogenase
  converts pyruvate to acetyl-CoA,
  which is fed into the TCA cycle.
  (The subject of the next chapter)

 This is only allowed under aerobic
 conditions. Anerobic growth results in
 fermentation, in which NADH is
 oxidized by the end-product of
 glycolysis.
      Reoxidation of NADH
There are 3 ways to deal with pyruvate:

2)         Lactate dehydrogenase
  uses NADH to reduce pyruvate to
  lactate.

 Pyruvate + NADH + H+ ® lactate +
 NAD+
      Reoxidation of NADH
There are 3 ways to deal with pyruvate:
3)   Many microorganisms use a 2-step
  process:

 Pyruvate decarboxylase uses TPP to
 catalyze: pyruvate ® acetaldehyde +
 CO2

 Alcohol dehydrogenase uses NADH
 to reduce acetaldehyde to ethanol.
  Use of other carbohydrates
• Dietary oligosaccharides,
  polysaccharides, and protein-linked
  carbohydrates are converted to
  monomers by the action of several
  hydrolase enzymes (dextrinases, maltase,
 lactase, sucrase, trehalase, etc.).
• This yields glucose, fructose, galactose,
  and mannose (+ others)
  Use of other carbohydrates
• Fructose:
  Hexokinase can phosphorylate Frc to Frc-6-
  P i.
• Mannose:
  Hexokinase can phosphorylate Man to Man-
  6-Pi. This is a substrate for
  phosphomannose isomerase, which
  converts it to Frc-6-Pi. (Remember that Man
  is the C-2 epimer of Glc, so the reaction is
  essentially the same as
  phosphohexoisomerase, but the enzyme
  has to be different to accommodate the
  alternate stereochemistry at C-2.)
  Use of other carbohydrates
• Galactose:
1. Phosphorylated to Gal-1-Pi by
   galactokinase.
2. This is a substrate for a uridyl
   transferase enzyme, which catalyzes:
  Gal-1-Pi + UDP-Glc ® UDP-Gal + Glc-1-Pi
– Then UDP-glucose 4-epimerase acts:
  UDP-Gal ® UDP-Glc         (uses tightly bound NAD)


  This gives overall transformation of Gal to Glc-1
     -Pi.
        Glycogen breakdown
The glucose in glycogen is mobilized by 2
  enzymes:

Glycogen phosphorylase
• Catalyzes the phosphorolysis of terminal glucose
  residues (at a non-reducing end) using inorganic
  phosphate:
  (a-1,4-Glc)n + Pi ® (a-1,4-Glc)n-1 + Glc-1-Pi

• Cannot cleave beyond a point 4 residues upstream of
  an
  a1®6 branch point.
        Glycogen breakdown
The glucose in glycogen is mobilized by 2
  enzymes:

Debranching enzyme
Removes branches by consecutive action of 2 activities:
• Transferase transfers the 3 terminal a1®4 linked
  residues from an a1®6 linkage to the end of the
  "parent" branch.
• a1®6 glucosidase cleaves the lone a1®6 linked
  glucose, leaving an unbranched chain.
         Glycogen synthesis
Glycogen synthesis is carried out by 2
 enzymes:
  1) UDP-glucose pyrophosphorylase
  Glc-1-Pi + UTP ® UDP-Glucose + PPi

    DG°' ≈ 0 for this reaction, but it is driven
    forward by pyrophosphatase: PPi ® 2 Pi

    (UDP-glucose is one of several sugar nucleotides
    used by the cell as high-energy sugar donors to
    synthesize polysaccharides.)
          Glycogen synthesis
Glycogen synthesis is carried out by 2
 enzymes:
   2)     Glycogen synthase
   UDP-Glc + glycogenn ® glycogenn+1 + UDP

     (The reaction mechanism may involve a
     carboxonium ion intermediate.)

UTP is rephosphorylated by NDP kinase:
   NDP + ATP ® NTP + ADP
Thus, the overall reaction is:
   Glc-1-Pi + ATP + glycogenn ® glycogenn+1 + ADP + 2
     P
         Glycogen synthesis
• The a1®6 branch points are created by
  "glycogen branching enzyme”
• It transfers a fragment of 6-7 glucose residues
  (~1 turn of an a1®4 glucose helix) from the end
  of one chain to the C-6 hydroxyl of a residue at
  least 3-4 residues upstream on the same chain.
           Glycogen synthesis
• Note that glycogen synthesis is initiated by a protein
  called glycogenin.
• The reducing end of glycogen is actually not a reducing
  end – it is connected by a glycosidic linkage to Tyr194 of
  glycogenin.

• Starch synthesis occurs very similarly in plants, except
  that the sugar nucleotide used is ADP-glucose, rather
  than UDP-glucose.
           Gluconeogenesis
• Overall conversion:
   2 pyruvate ® glucose
   driven by hydrolysis of 6 ATPs
• Essentially, this is the reverse of glycolysis.
  However, not all of the reactions are the same.
  The ones outside of the pools that are highly
  exergonic are different.
• The reactions between pools 2 and 3 are not
  highly exergonic under physiological conditions,
  and can thus be employed in either direction.
             Gluconeogenesis
Pyruvate ® PEP
1)       Pyruvate carboxylase
     Carboxylates pyruvate to oxaloacetate (OAA) in
     a reaction that uses biotin and requires ATP.

2)       PEP carboxykinase
     Decarboxylates OAA with concomitant transfer
     of phosphate from GTP(ATP) to the oxygen on
     C-2.
     (b-decarboxylation of OAA is very favorable,
     helping to drive the reaction.)
               Gluconeogenesis
Pyruvate ® PEP
1)       Pyruvate carboxylase
     Carboxylates pyruvate to oxaloacetate (OAA) in a
     reaction that uses biotin and requires ATP.

2)       PEP carboxykinase
     Decarboxylates OAA with concomitant transfer of
     phosphate from GTP(ATP) to the oxygen on C-2.
     (b-decarboxylation of OAA is very favorable, helping to
     drive the reaction.)
            Gluconeogenesis
Pyruvate ® PEP
• Complication: These enzymes are often in the
  mitochondrion. In this case, pyruvate is
  imported into the mitochondrion and PEP is
  exported.
• If PEP carboxykinase is in the cytosol, reducing
  power can be exported in the form of malate
  (TCA cycle intermediate).
• Pyruvate is imported into the mitochondrion,
  where the decision is made:
  – either feed it into the TCA cycle (as acetyl-CoA)
  – or convert it to PEP (for gluconeogenesis).
         Gluconeogenesis
PEP ® ® ® Frc-1,6-bisPi
• These reactions are the same as in
  glycolysis.

Frc-1,6-bisPi ® Frc-6-Pi
• Fructose bisphosphatase catalyzes:
  Frc-1,6-Pi + H2O –> Frc-6-Pi + Pi
           Gluconeogenesis
Glc-6-Pi ® Glc
• glucose-6-phosphatase catalyzes:
  Glc-6-Pi + H2O ® Glc + Pi

• However, the objective of gluconeogenesis in
  most cells is to store carbohydrate, not to make
  free glucose. Thus, hexose monophosphates
  will be used, and glucose-6-phosphatase is
  usually not present.
• The exception is in the liver, which plays a key
  role in maintaining blood glucose levels. Free
  glucose can be exported into the blood.

				
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