This study ascribes the large steady-state D2O isotope effect on kcat of pyruvate kinase (PEP + ADP----pyruvate + ATP) to the reprotonation of the product form of the enzyme for use in forming pyruvate. Previous tritium trapping experiments [Rose, I. A., & Kuo, D. J. (1989) Biochemistry 28, 9579-9585] with muscle pyruvate kinase showed that the proton used for ketonization of enolpyruvate is derived from an enzyme "pool" that contains three kinetically equivalent hydrogens that could be trapped in a nontritiated "chase" medium by high levels of ADP and PEP. The exchange of this pool with the medium was rapid in the free enzyme (approximately 1400 s-1), prior to addition of PEP, and apparently much less in the completed complex. The dissociation rate constant was determined by using the dissociation-competition equation koffT = K1/2kcat/Km, where kcat/Km is the steady-state parameter for PEP and K1/2 is the concentration of PEP required to trap 50% of the isotope that could be trapped. The present study shows that the competition constant, K1/2, is decreased by approximately 5-fold in D2O, the same effect see on kcat under conditions where kcat/Km, measured in the steady state, is not changed. The common effect of D2O on kcat in the steady state and koffT in pulse/chase suggests that the forward reaction rate is determined by hydrogen transfer to the enzyme. Further evidence indicates that the kinetically important proton in question is the proton used for ketonization of enolpyruvate, the substrate proton.(ABSTRACT TRUNCATED AT 250 WORDS)