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

Regulation of glycolysis and gluconeogenesis

Phosphofructokinase (PFK)

Fructose 6P + ATP 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
  • inhibitory action of ATP is reversed by AMP

PFK activity increases when 

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
  • 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 fructose2,6- bisphosphatase (FBPase-2)

in starved rat, <1M in liver

in well-fed rat, 20M in liver

fructose 6Pfructose 2,6-bisP

  • 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

 [F6P]  [F2,6bisP] PFK

feedforward stimulation

  1. covalent modification

phosphorylation by cAMP-dependent protein kinase A (PKA)

at Ser residue

dephosphorylation by phosphoprotein phosphatase

phosphorylation inhibitsPFK-2 activity and activatesFBPase-2 activity

dephosphorylation

PFK-2

FBPase-2

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

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

  • Catalytic properties of L type (but not M) controlled by reversible phosphorylation

 [glucose]

 glucagon

 cAMP cascade

PK activity

 GYLCOLYSIS

Pyruvate carboxylase

  • Activated by acetyl CoA
  • Inhibited by ADP

Phosphoenolpyruvate carboxykinase

  • Inhibited by ADP

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