Pyruvate kinase: is the mechanism of phospho transfer associative or dissociative?

Abstract

To test for the possibility that pyruvate kinase proceeds via a dissociative path, we have investigated whether the complex enzyme . ADP . metaphosphate is transiently formed from the complex enzyme . ATP. It is shown that when highly purified pyruvate kinase is used, the rate of positional oxygen isotope exchange in ATP (beta, gamma bridge and beta nonbridge) is about 10(4) times slower in the absence of the cosubstrate pyruvate than it is in the presence of pyruvate. Further, the rate of racemization of the gamma-phospho group of [gamma (S)-16O,17O,18O]-ATP is undetectable, being at least 30 times slower even than the rate of positional isotope exchange. These tests thus provide no evidence that pyruvate kinase follows a dissociative mechanism. Indeed, it is argued that the available data are more consistent with an associative path. Evidence is presented that the single, associative, transition state is symmetrical, in which bond making and bond breaking processes are rather precisely balanced.