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

Rate constants for formation of acetaldehyde from pyruvate catalyzed b y the thiamin diphosphate (TDP)-dependent enzyme pyruvate decarboxylas e (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 pyruvam ide, 10 mM MgSO4, and 12.5 muM sodium pyruvate. Observed rate constant s in the range k(obsd) = 2.5-6.7 s-1 for 33-104 muM (8-15 mg mL-1) pyr uvamide-activated PDC agree with values of k(obsd) calculated by numer ical integration with microscopic rate constants derived previously fr om steady-state kinetic isotope effects. The observed rate constant k( obsd) = 6.7 +/- 0.4 s-1 is independent of the concentration of pyruvam ide-activated PDC in the range 104-150 muM. The decrease in the concen tration dependence of the observed rate constants at > 104 muM PDC is consistent with either a change in rate-limiting step or complex forma tion involving the reactants. There is little or no primary kinetic is otope effect, (k(H)/k(D))obsd less-than-or-equal-to 1.2, for C(2)-hydr on exchange from PDC-bound TDP for 33-104 muM pyruvamide-activated PDC . This provides evidence against rate-limiting C(2)-proton transfer be tween C(2)-H in PDC-bound TDP and a catalytic base with -7 less-than-o r-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.