The Correlation of Reaction Kinetics and Substrate Binding with the Mechanism of Pyruvate Kinase

Abstract

Applications of isotopic techniques have contributed considerably to the understanding of kinase mechanisms. Studies with 018 (l-3) have shown that various kinase reactions proceed by transfer of a phosphoryl group without exchange of the phosphoryl oxygens with water or substrates’ Evidence that the kinase reactions do not involve formation of a phosphoryl enzyme intermediate is given by the inability of pyruvate kinase to catalyze an exchange of pyruvate-Cl4 with phosphoenolpyruvate (1,2) and of acetate kinase to catalyze exchange of acetateCl4 with acetyl phosphate (4) in the absence of adenine nucleotides. A phosphoryl enzyme intermediate has been suggested for the hexokinase reaction (5), but the findings of more recent experiments with hexokinase as well as with pyruvate kinase argue strongly against this possibility (6). These and the previously mentioned results support the suggestion made earlier (l-3, 7) that the kinase reactions proceed by a direct transfer2 of a phosphoryl group from the donor to the acceptor. Kinetic and binding studies with pyruvate kinase should reveal if substrate interactions occur in a manner consistent with a direct transfer mechanism, and it is the principal purpose of this paper to report the results of such studies. In a direct transfer mechanism, adenosine diphosphate and adenosine triphosphate would be expected to share one binding site, and pyruvate and phosphoenolpyruvate another binding site on the enzyme, with the further provision that the binding occur so that the transferable phosphoryl group of adenosine triphosphate or of phosphoenolpyruvate occupies a common position on the enzyme. Biicher has suggested, however, that in catalysis by $phosphoglycerate kinase, the adenosine triphosphate occupies a different site than adenosine diphosphate on the enzyme