Escherichia coli phosphoenolpyruvate carboxylase. Studies on the mechanism of synergistic activation by nucleotides.
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.
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D002262:Carboxy-Lyases / Q000378:metabolism*; D003565:Cytidine Diphosphate / Q000494:pharmacology*; D003597:Cytosine Nucleotides / Q000494:pharmacology*; D004357:Drug Synergism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism*
D003597:Cytosine Nucleotides; D003565:Cytidine Diphosphate; D000105:Acetyl Coenzyme A; D006160:Guanosine Triphosphate; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase; D008274:Magnesium