J. Biol. Chem., Vol. 255, Issue 4, 1635-1642, Feb, 1980
Escherichia coli phosphoenolpyruvate carboxylase. Studies on the mechanism of synergistic activation by nucleotides
TE Smith, KA Balasubramanian and A Beezley
Kinetic studies were done to obtain a quantitative estimation of the
synergistic interactions that occur between phosphoenolpyruvate carboxylase
(orthophosphate:oxaloacetate carboxylase (phosphorylating) E.C. 4.1.1.31)
from Escherichia coli K12 and various combinations of its primary
substrate, P-enolpyruvate, and the activators acetylcoenzyme A, CDP, GTP,
and fructose 1,6-bisphosphate. The analysis involves the evaluation of
apparent K values, KS for P-enolpyru;ate and KA for activators, as a
function of the concentration of P-enolpyruvate or another activator in the
case of KA determinations. Methods are presented which allow the
determination of dissociation constants for P- enolpyruvate and activators
from binary, ternary, and quaternary complexes of enzyme with substrates or
activators, or both. It was observed that synergistic activation occurs
with acetyl coenzyme A and all of the coactivators but not with fructose
1,6-bisphosphate and the other co-activators. The enhancement of binding
from the binary enzyme substrate (or activator) complex to the ternary or
quaternary complexes is in the range of 100-fold. The dissociation
constants for P- enolpyruvate, acetyl coenzyme A, CDP, and fructose
1,6-bisphosphate are the same from any active enzyme species. Synergistic
activation by multiple activators reflects the ability of co-activators to
shift the equilibrium concentrations of active enzyme species away from the
inactive forms via a modified "cascade" scheme, thus decreasing the
probability that dissociation of any one activator will yield an inactive
enzyme species.
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