SINGLE-TURNOVER STUDIES ON BREWERS-YEAST PYRUVATE DECARBOXYLASE - C(2)-PROTON TRANSFER FROM THIAMIN DIPHOSPHATE

Rate constants for formation of acetaldehyde from pyruvate catalyzed by the thiamin diphosphate (TDP)-dependent enzyme pyruvate decarboxylase (PDC; EC 4.1.1.1) from Saccharomyces carlsbergensis were determined under single-turnover conditions at 30-degrees-C in 100 mM sodium 2-(N-morpholino)ethanesulfonate buffer (pH 6.00) containing 100 mM pyruvamide, 10 mM MgSO4, and 12.5 muM sodium pyruvate. Observed rate constants in the range k(obsd) = 2.5-6.7 s-1 for 33-104 muM (8-15 mg mL-1) pyruvamide-activated PDC agree with values of k(obsd) calculated by numerical integration with microscopic rate constants derived previously from steady-state kinetic isotope effects. The observed rate constant k(obsd) = 6.7 +/- 0.4 s-1 is independent of the concentration of pyruvamide-activated PDC in the range 104-150 muM. The decrease in the concentration dependence of the observed rate constants at > 104 muM PDC is consistent with either a change in rate-limiting step or complex formation involving the reactants. There is little or no primary kinetic isotope effect, (k(H)/k(D))obsd less-than-or-equal-to 1.2, for C(2)-hydron exchange from PDC-bound TDP for 33-104 muM pyruvamide-activated PDC. This provides evidence against rate-limiting C(2)-proton transfer between C(2)-H in PDC-bound TDP and a catalytic base with -7 less-than-or-equal-to DELTApK(a) (= pK(a)BH - pK(a)C(2)H) less-than-or-equal-to 7 to form a discrete ylide intermediate during catalysis by PDC.