Biochemistry 432/832

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Biochemistry 432/832

• September 03
• Chapter 23 GG
• Gluconeogenesis
• Glycogen metabolism

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Announcements -
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Comparison of glycolysis and gluconeogenesis
pathways
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Energetics of Glycolysis

• The elegant evidence of regulation!
• ?G in cells is revealing
• Most values near zero
• 3 of 10 reactions have large, negative ? G
• Large negative ? G reactions are sites of
  regulation!
• Reactions 1, 3 and 10 should be different to go
  into opposite direction

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Gluconeogenesis

• Something Borrowed, Something New
• Seven steps of glycolysis are retained
• Steps 2 and 4-9
• Three steps are replaced
• Steps 1, 3, and 10 (the regulated steps!)
• The new reactions provide for a spontaneous
  pathway (?G negative in the direction of sugar
  synthesis), and they provide new mechanisms of
  regulation

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Pyruvate Carboxylase

• Pyruvate is converted to oxaloacetate
• The reaction requires ATP and bicarbonate as
  substrates
• Biotin-dependent
• Biotin is covalently linked to an active site
  lysine
• Acetyl-CoA is an allosteric activator
• Regulation when ATP or acetyl-CoA are high,
  pyruvate enters gluconeogenesis
• The "conversion problem" in mitochondria

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The pyruvate carboxylase reaction
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Linkage of biotin to lysine residue in pyruvate
carboxylase
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Pyruvate carboxylase is a compartmentalized enzyme
Oxaloacetate is formed in mitochondria It cannot
be transported to the cytosol
It is converted to malate in mitochondria and
back to oxaloacetate in the cytosol
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PEP Carboxykinase

• Conversion of oxaloacetate to PEP
• Lots of energy needed to drive this reaction!
• Energy is provided in 2 ways
• Decarboxylation is a favorable reaction
• GTP is hydrolyzed
• GTP used here is equivalent to an ATP

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The PEP carboxykinase reaction
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Fructose-1,6-bisphosphatase

• Hydrolysis of F-1,6-P to F-6-P
• Thermodynamically favorable - ?G in liver is -8.6
  kJ/mol
• Allosteric regulation
• citrate stimulates
• fructose-2,6-bisphosphate inhibits
• AMP inhibits

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The fructose-1,6-biphosphatase reaction
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Glucose-6-Phosphatase

• Conversion of Glucose-6-P to Glucose
• Presence of G-6-Pase in ER of liver and kidney
  cells makes gluconeogenesis possible
• Muscle and brain do not do gluconeogenesis
• G-6-P is hydrolyzed as it passes into the ER
• ER vesicles filled with glucose diffuse to the
  plasma membrane, fuse with it and open, releasing
  glucose into the bloodstream

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Glucose-6-phosphatase is localized in the ER
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Lactate Recycling

• How your liver helps you during exercise....
• Vigorous exercise can lead to a buildup of
  lactate and NADH, due to oxygen shortage and the
  need for more glycolysis
• NADH can be reoxidized during the reduction of
  pyruvate to lactate
• Lactate is then returned to the liver, where it
  can be reoxidized to pyruvate by liver LDH
• Liver provides glucose to muscle for exercise and
  then reprocesses lactate into new glucose

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The Cori Cycle
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Gerty and Carl Cori
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Cori Cycles
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Regulation of Gluconeogenesis

• Reciprocal control with glycolysis
• When glycolysis is turned on, gluconeogenesis
  should be turned off
• When energy status of cell is high, glycolysis
  should be off and pyruvate, etc., should be used
  for synthesis and storage of glucose
• When energy status is low, glucose should be
  rapidly degraded to provide energy
• The regulated steps of glycolysis are the very
  steps that are regulated in the reverse direction!

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Regulation of glycolysis and gluconeogenesis
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Gluconeogenesis Regulation II

• Allosteric and Substrate-Level Control
• Glucose-6-phosphatase is under substrate-level
  control, not allosteric control
• The fate of pyruvate depends on acetyl-CoA
• F-1,6-bisPase is inhibited by AMP, activated by
  citrate - the reverse of glycolysis
• Fructose-2,6-bisP is an allosteric inhibitor of
  F-1,6-bisPase

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Inhibition of fructose-1,6-bisphosphatase by
fructose-2,6-bisphosphate - synergistic effect of
F-2,6-P and AMP
F-2,6-P
F-2,6-P
AMP
25 mM AMP
No AMP
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Synthesis and degradation of F-2,6,-bisP are
catalyzed by the same enzyme
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Substrate cycles
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Substrate cycles

• Simultaneous activity of Phosphofructokinase
  (glycolysis) and F-1,6-bisPase (gluconeogenesis)
  yields a substrate cycle
• Reverse reaction decreases steady state flux
  through the pathway
• Could explain how 10 change in ATP concentration
  results in 90-fold increase in the flux through
  glycolysis
• Synergistic Fructose-2,6-bisP / AMP / ATP /
  citrate (NAD/NADH ratio, glucose-6-P, pyruvate,
  etc.) regulation provide alternative explanation

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Substrate cycles

• Three potential substrate cycles in glycolysis
  and gluconeogenesis
• Example of Phosphofructokinase (glycolysis) and
  F-1,6-bisPase (gluconeogenesis)
• Reciprocal regulation does not work at high
  F-1,6-P
• Perhaps substrate cycling occurs only at high
  concentrations of F-1,6-P (PFK product) - - this
  prevents accumulation of excessively high levels
  of F-1,6-P

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Glucose
Glucose-6-P
Glycogen
Ribose-5-P NADPH
Fructose-6-P
Reducing power
Nucleic acid synthesis
Glyceraldehyde-3-P
Pyruvate
ATP
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23.3 Glycogen Catabolism

• Getting glucose from storage (or diet)
• Glycogen is a storage form of glucose
• ?-Amylase is an endoglycosidase
• It cleaves amylopectin or glycogen to maltose,
  maltotriose and other small oligosaccharides
• It is active on either side of a branch point,
  but activity is reduced near the branch points
• Debranching enzyme cleaves "limit dextrins"
• The 2 activities of the debranching enzyme

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Hydrolysis of glycogen by amylases
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The reactions of glycogen debranching enzyme. 1)
Transfer of 3 glucose residues to another branch
and 2) cleavage of a single glucose residue at
the branch point
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Metabolism of Tissue Glycogen

• Digestive breakdown is unregulated - 100!
• But tissue glycogen is an important energy
  reservoir - its breakdown is carefully controlled
  
• Glycogen consists of "granules" of high MW
• Glycogen phosphorylase cleaves glucose from ends
  of glycogen molecules
• This is a phosphorolysis, not a hydrolysis
• Metabolic advantage product is a sugar-P - a
  "sort-of" glycolysis substrate

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The glycogen phosphorylase reaction -
phosphorolysis
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Glycogen Phosphorylase

• A beautiful protein structure!
• A dimer of identical subunits (842 res. each)
• Each subunit contains a PLP, which participates
  in phosphorolysis
• Chapter 15

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Dimer
Monomer