The Proton Transfer Reactions of Muscle Pyruvate Kinase

Rabbit muscle pyruvate kinase detritiates phosphoenolpyruvate 3-tritium under conditions of the net forward reaction prior to the release of pyruvate. This exchange requires that release of pyruvate is not rapid with respect to reversal of the proton transfer steps involved in the catalysis of its enolization. This conclusion is consistent with the low tritium isotope effect that is observed for the hydrogen exchange on pyruvate that is activated by ATP. An intrinsic isotope effect for the proton abstraction step as high as 26 can be expected since such high values were observed in the enzymatic enolization of pyruvate activated by other phosphate compounds. Considerable exchange of tritium from tritiated water into remaining phosphoenolpyruvate during the course of the forward pyruvate kinase reaction is observed, indicating that the enzyme-bound pyruvate-ATP complex that is generated in the forward reaction can return to substrate form at a significant rate relative to product release. A kinetic analysis indicates that the release of phosphoenolpyruvate and ADP may be a rate-determining factor in this exchange rate. Thus, the conclusions of earlier workers that muscle pyruvate kinase follows rapid equilibrium kinetics require re-examination. The effects of varying pH, mono- and divalent cations, temperature, and alternative activators on the rates of proton exchange are reported.