Chem 356 Structure and Function in Biochemistry
Lecture Oct 27-30
Allosteric Control of Glycolysis and Gluconeogenesis
 glycolytic pathway
degrades glucose  ATP
provides building blocks
 rate of conversion of glucose into pyruvate is
regulated to meet these two cellular needs
In metabolic pathways, enzymes catalyzing irreversible
reactions are potential sites of control
Glycolysis—irreversible reactions
 hexokinase
 phosphofructokinase
 pyruvate kinase
 each reaction serves as a control site
 activities are controlled by allosteric effectors, or by
covalent modification, or by transcriptional control
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Regulation of glycolysis and gluconeogenesis
glucose
hexokinase
G6phosphatase
Glucose 6-phosphate
Fructose 6-phosphate
PFK
F1,6bisPase
Fructose 1,6-bisphosphate
phosphoenolpyruvate
PEP carboxykinase
Pyruvate
kinase
oxaloacetate
Pyruvate
carboxylase
pyruvate
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Phosphofructokinase (PFK)
Fructose 6P + ATP
PFK
fructose 1,6-bisP + ADP
 most important control element in glycolytic
pathway
 340 kDa tetrameric enzyme, allosteric
 inhibited by high levels of ATP
 negative heterotropic effector
 2 conformational states
T  R
in equilibrium
 each subunit has 2 binding sites for ATP
 substrate site
 regulatory site
 substrate site binds ATP in either state
 inhibitor site binds ATP in T state
 other substrate, fructose 6P, binds to R state
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 inhibitory action of ATP is reversed by AMP
ATP
 PFK activity increases when

AMP
 Glycolysis stimulated as energy charge
falls
 Citrate enhances inhibitory effect of ATP
 High citrate levels indicate abundance of
biosynthetic precursors
 Stop glucose degradation via
glycolysis
Fructose 1,6-bisphosphatase (F1,6BPase)
 Inhibited by AMP
 Activated by citrate
Fructose 2,6-bisphosphate
 Potent allosteric regulator of gluconeogenesis
and glycolysis
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 1980 – F2,6-bisP activator of PFK
 1981 –F2,6-bisP inhibitor of F1,6-BPase
 [F2,6-bisP] in the cell depends on
 rate of synthesis by
phosphofructokinase-2 (PFK-2)
 rate of degradation by fructose2,6bisphosphatase (FBPase-2)
 in starved rat, <1M in liver
 in well-fed rat, 20M in liver
PFK-2
fructose 6P
fructose 2,6-bisP
FBPase-2
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 the 2 enzymatic activities on one protein
 bifunctional (or tandem) enzyme
 the opposing activities of this
bifunctional enzyme are regulated in 2
ways
 phosphorylation vs
dephosphorylation
1. F6P (substrate of PFK and product of
F1,6BPase) allosterically activates PFK-2 and
inhibits FBP-2ase
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 [F6P]
 [F2,6bisP]
 PFK
feedforward stimulation
2. covalent modification
 phosphorylation by cAMP-dependent
protein kinase A (PKA)
 at Ser residue
 dephosphorylation by phosphoprotein
phosphatase
 phosphorylation inhibits PFK-2 activity
and activates FBPase-2 activity
 dephosphorylation
 PFK-2
 FBPase-2
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Hexokinase
 inhibited by G6P
 if PFK inactive then F6P
 F6P then G6P
 inhibition of PFK inhibits hexokinase
However
 liver possesses glucokinase an isoform of
hexokinase
 glucokinase not inhibited by G6P
 phosphorylates glucose only when it is
abundant because >> KM than hexokinase
 5 mM glucokinase; 0.1 mM hexokinase
 role of glucokinase is to provide G6P for the
synthesis of glycogen
 high KM of glucokinase in liver gives
brain and muscle priority for glucose when
limited
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Pyruvate kinase (PyK)
 tetramer of 57 KDa subunits (228 KDa)
 catalyzes the third irreversible step in
glycolysis
 controls product outflow from pathway
 pyruvate  building block or oxidized
(ATP)
 exists as isoforms
 L-type: liver
 M-type: muscle and brain
 allosterically inhibited by
 ATP
 Alanine
 Acetyl CoA
 allosterically activated by
 F1,6-bisP
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 Catalytic properties of L type (but not M)
controlled by reversible phosphorylation
 [glucose]
 glucagon
 cAMP cascade
phosphorylation
of pyruvate kinase
 PK activity
 GYLCOLYSIS
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Pyruvate carboxylase
 Activated by acetyl CoA
 Inhibited by ADP
Phosphoenolpyruvate carboxykinase
 Inhibited by ADP
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