GLYCOLYSIS

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GLYCOLYSIS

• Reading
• Harpers Biochemistry pp. 190-198
• Lehninger Principles of Biochemistry 3rd Ed.
  pp. 527-566
• Glycolysis- from the Greek-
• glykys- sweet
• lysis- splitting

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OBJECTIVES

• To understand how the glycolytic pathway is used
  to convert glucose to pyruvate (and lactate) with
  conservation of chemical potential energy in the
  form of ATP and NADH.
• To learn the intermediates, enzymes, and
  cofactors of the glycolytic pathway.

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Major pathways of glucose utilization in cells of
higher plants and animals

• Although not the only possible fates for glucose,
  these three pathways are the most significant in
  terms of the amount of glucose that flows through
  them in most cells.

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• Glucose is the major fuel of most organisms.
• It is relatively rich in potential energy-
  complete oxidation to CO2 and H2O proceeds with a
  free-energy change of -2,840 kJ/mol.
• By storing glucose as high molecular weight
  polymers (starch/glycogen) a cell can stockpile
  large quantities of hexose units.
• When energy demands increase, glucose can be
  released quickly from storage and used to produce
  ATP either aerobically or anaerobically.

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• Glycolysis occurs in the cytosol of cells, is
  common to most organisms, and in humans occurs in
  virtually all tissues.
• Most tissues have at least a minimal requirement
  for glucose. In the brain, the requirement for
  glucose is substantial, in erythrocytes, it is
  nearly total.
• In glycolysis, a molecule of glucose is degraded
  in a series of steps catalyzed by ten cytosolic
  enzymes, to yield two molecules of the 3 carbon
  compound, pyruvate.
• During those sequential reactions, some of the
  free energy released is conserved in the form of
  ATP and NADH

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Biomedical Importance

• Of crucial biomedical significance is the ability
  of glycolysis to provide ATP in the absence of
  oxygen.
• This allows skeletal muscle to perform at high
  levels when aerobic oxidation becomes
  insufficient, and allows cells to survive anoxic
  episodes.
• Diseases associated with impaired glycolysis
• ?Hemolytic anemia
• - of the defects in glycolysis that cause
  hemolytic anemia, pyruvate kinase deficiency
  (genetic mutations) is the most common.
• - mature erythrocytes contain no mitochondria,
  totally dependent upon glycolysis for ATP.
• - ATP is required for Na/K-ATPase-ion transport
  system which maintain the proper shape of the
  erythrocyte membrane.
• ?Lactic Acidosis
• - can be due to several causes of improper
  utilization of lactate.

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Glycolysis is only the first step in the
degradation of glucose

• Three possible catabolic fates of the pyruvate
  formed in glycolysis. Pyruvate also serves as a
  precursor in many anabolic reactions, not shown
  here.

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Where glycolysis fits in the big picture of
catabolism
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Overview of glycolysis
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Energy Transformations during Glycolysis

• Glucose 2 NAD 2 ADP 2 Pi ? 2 Pyruvate 2
  NADH 2 H 2 ATP H2O
• (Note Much, 95, of the energy remains in
  pyruvate)
• Resolve into two processes
• Glucose 2 NAD ? 2 pyruvate 2 NADH 2 H
• ?Go1 -146 kJ/mol
• 2 ADP 2 Pi ? 2 ATP 2 H2O
• ?Go 2(30.5 kJ/mol) 61 kJ/mol
• Overall free energy change -146 61 -85
  kJ/mol

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Why phosphorylated intermediates?

• Each of the nine glycolytic intermediates between
  glucose and pyruvate is phosphorylated
• 1. Phosphate groups are ionized at pH 7, giving
  each glycolytic intermediate a net negative
  charge. Because the plasma membrane is
  impermeable to charged molecules, the
  phosphorylated intermediates cannot disperse out
  of the cell.
• 2. Energy used in the formation of the
  phosphate ester is partially conserved. High
  energy phosphate compounds formed in glycolysis
  (1,3-bisphosphoglycerate and phosphoenolpyruvate)
  donate phosphoryl groups to ADP to form ATP.
• 3. Binding energy resulting from the binding of
  phosphate groups to the active sites of enzymes
  lowers the activation energy and increases the
  specificity of the enzymatic reactions.

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Step 1 Phosphorylation of glucose

• Glucose is activated by phosphorylation at C6
• Reaction is catalyzed by hexokinase, present in
  virtually all extrahepatic cells. Has high
  affinity (low Km) for glucose, so phosphorylates
  essentially all the glucose that enters cell,
  maintaining a large glucose gradient. Will also
  phosphorylate other hexose sugars. Under
  physiological conditions, reaction is essentially
  irreversible.
• In liver, glucose is phosphorylated by
  glucokinase. This enzyme has a low affinity
  (high Km) for glucose, is specific for glucose,
  and its main task is to remove glucose from blood
  following a meal.

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Step 2 Conversion of glucose 6-phosphate to
fructose 6-phosphate

• The enzyme phosphohexose isomerase catalyzes the
  reversible isomerization of glucose 6-phosphate
  (an aldose) to fructose 6-phosphate (a ketose)
• As predicted for the relatively small change in
  standard free energy, the reaction proceeds
  readily in either direction, and requires Mg2

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Step 3 Phosphorylation of fructose 6-phosphate
to fructose 1, 6-bisphosphate

• Phosphofructokinase-1 catalyzes the transfer of a
  phosphoryl group from ATP to fructose 6-phosphate
  to yield fructose 1, 6-bisphosphate.
• This reaction is essentially irreversible under
  cellular conditions.
• Phosphofructokinase-1 is a regulated enzyme at a
  major point in the regulation of glycolysis.
• PFK-1 activity is increased whenever the cells
  ATP supply is depleted or when ADP/Pi are in
  excess.
• Activity is inhibited whenever the cell has ample
  ATP and is well supplied by other fuels such as
  fatty acids.

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Step 4 Cleavage of fructose 1, 6-bisphosphate

• The enzyme fructose 1, 6-bisphosphate aldolase,
  called just aldolase, catalyzes the cleavage of
  fructose 1, 6-bisphosphate into two different
  triose phosphate, glyceraldehyde 3-phosphate and
  dihydroxyacetone phosphate
• In cells, this reaction can proceed in either
  direction, and proceeds to the right during
  glycolysis because products are quickly removed.

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Step 5 Interconverstion of the triose phosphates

• Dihydroxyacetone phosphate is rapidly and
  reversibly converted to glyceraldehyde
  3-phosphate by triose phosphate isomerase
• The C-1, C-2, C-3 of the starting glucose now
  become chemically indistinguishable for the C-6,
  C-5, and C-4, respectively.
• This reaction completes the prepatory phase of
  glycolysis
• Other hexoses (fructose, mannose, galactose) can
  also be converted into glyceraldehyde 3-phosphate

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The payoff phase of glycolysis producing ATP
NADH

• 2 molecules of glyceraldehyde 3-phosphate ? 2
  molecules of pyruvate
• Step 6 Glyceraldehyde 3-phosphate dehydrogenase
  catalyzes the oxidation of glyceraldehyde
  3-phosphate to 1, 3-bisphosphoglycerate
• Note that the aldehyde group is dehydrogenated to
  an acyl phosphate, which has a very high standard
  free energy of hydrolysis (-49.3 kJ/mol).
• Glyceraldehyde 3-phosphate dehydrogenase is
  inhibited by iodoacetetate

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Step 7 Phosphoryl transfer from 1,
3-bisphospho-glycerate to ADP

• The enzyme phosphoglycerate kinase transfers the
  high-energy phosphoryl group from the carboxyl
  group to ADP, forming ATP and 3-phosphoglycerate.
• Steps 6 and 7 represent an energy-coupling
  process in which 1, 3-phosphoglycerate is the
  common intermediate.
• Glyceraldehyde 3-phosphate ADP Pi NAD
  
• 3-phosphoglycerate ATP NADH H
• ?Go1 -12.5 kJ/mol

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Step 8 Conversion of 3-phosphoglycerate to
2-phosphoglycerate

• The enzyme phosphoglycerate mutase catalyzes a
  reversible shift of the phosphoryl group between
  C-2 and C-3 of glycerate. Mg2 is essential

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Step 9 Dehydration of 2-phosphoglycerate to
phosphoenolpyruvate

• Enolase promotes reversible removal of a molecule
  of water from 2-phosphoglycerate to yield
  phosphoenolpyruvate
• Standard free energy of hydrolysis of the
  phosphate groups of the reactant and product are
  -17.6 kJ/mol and -61.9 kJ/mol, respectively.
• ie. The loss of the water molecule causes a
  redistribution of energy within the molecule,
  generating a super high-energy phosphate compound.

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Step 10 Transfer of the phosphoryl group from
phosphoenolpyruvate to ADP

• This last step in glycolysis is catalyzed by
  pyruvate kinase, which requires K and Mg 2 or
  Mn 2
• This step is also an important site of regulation
  
• The product pyruvate undergoes tautomerization
  from its enol to keto form which is more stable
  at pH 7

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Overall balance sheet - net gain of ATP

• Glucose 2 ATP 2 NAD 4 ADP 2 Pi ?
• 2 Pyruvate 2 ADP 2 NADH 2 H 4
  ATP 2 H2O
• or Glucose 2 NAD 2 ADP 2 Pi ?
• 2 Pyruvate 2 NADH 2 H 2 ATP 2
  H2O
• Under aerobic conditions, the two molecules of
  NADH are reoxidized to NAD by transfer of their
  electrons to the respiratory chain in the
  mitochondrion
• 2 NADH 2 H O2 ? 2 NAD 2 H2O
• During glycolysis
• Carbon pathway - Glucose ? 2x pyruvate
• Phosphate pathway - 2 ADP 2 Pi ? 2 ATP
• Electron pathway - Four electrons (two hydride
  ions) are transferred from 2 molecules
  of glyceraldehyde 3-phosphate to two of
  NAD

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Conversion of pyruvate to lactate

• Under hypoxic or anaerobic conditions, NADH
  generated by glycolysis cannot be reoxidized by
  O2 - NAD is required during glycolysis as
  electron acceptor in step 6.
• In these cases, NAD is regenerated from NADH by
  the reduction of pyruvate to lactate, catalyzed
  by lactate dehydrogenase. This allows glycolysis
  to occur in the absence of oxygen
• Lactate produced in muscle during a short burst
  of physical activity is converted back to glucose
  in the liver.

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Glycolysis is regulated at 3 steps involving non
equilibrium reactions

• Step 1 hexokinase
• glucose ? glucose 6-phosphate
• Step 3 phosphofructokinase
• fructose 6-phosphate ? fructose 1,
  6-bisphosphate
• Step 10 pyruvate kinase
• phosphoenolpyruvate ? pyruvate
• These are all exergonic and physiologically
  irreversible
• These enzymes function as valves, regulating
  the flow of carbon through glycolysis. The rates
  of these steps are limited not by the substrate
  but by the activity of the enzymes.
• Enzymes that catalyze these exergonic,
  rate-limiting steps are commonly the targets of
  metabolic regulation.
• Examples of regulation
• Phosphofructokinase-1 - inhibited by high levels
  of ATP. ATP binds to an allosteric site and
  lowers affinity for fructose 6-phosphate
• Hexokinase - allosterically inhibited by its
  product.
• Pyruvate kinase - inhibited by ATP

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Regulation of the flux through a multistep pathway

• Regulation occurs at steps that are
  enzyme-limited. At each of these steps (orange
  arrows), which are generally exergonic, the
  substrate is not in equilibrium with the product
  because the reaction is relatively slow the
  substrate tends to accumulate, just as river
  water accumulates behind a dam. In the
  substrate-limited reactions (blue arrows), the
  substrate and product are essentially at their
  equilibrium concentrations. In the steady state,
  all reactions in the sequence occur at the same
  rate, which is determined by the rate-limiting
  step.

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Feeder pathways for glycolysis
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SUMMARY

• Glycolysis is a universal metabolic pathway for
  the catabolism of glucose to pyruvate accompanied
  by the formation of ATP.
• The process is catalyzed by 10 cytosolic enzymes
  and there is a net gain of two ATPs per molecule
  of glucose.
• The NADH formed must be recycled to regenerate
  NAD. Under aerobic conditions this occurs
  during mitochondrial respiration under anaerobic
  conditions, NAD is regenerated by the conversion
  of pyruvate to lactate. Other organisms such as
  yeast regenerate NAD by reducing pyruvate to
  ethanol CO2 (fermentation)
• A variety of D-hexoses, including fructose,
  mannose, and galactose, can be funneled into
  glycolysis.
• Enzyme limited, regulated steps are catalyzed by
  hexokinase, phosphofructokinase-1, and pyruvate
  kinase.