Single-turnover studies on brewer's yeast pyruvate decarboxylase: C(2)-proton transfer from thiamin diphosphate

Article abstract of DOI:10.1021/ja00073a004

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°C in 100 mM sodium 2-(N-morpholino)ethanesulfonate buffer (pH 6.00) containing 100 mM pyruvamide, 10 mM MgSO₄, and 12.5 μM sodium pyruvate. Observed rate constants in the range k_obsd = 2.5-6.7 s^-1 for 33-104 μM (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 μM. The decrease in the concentration dependence of the observed rate constants at > 104 μM 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 ≤ 1.2, for C(2)-hydron exchange from PDC-bound TDP for 33-104 μM 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 ≤ ΔpK_a (= pK_a^BH - pK_a^C(2)H) ≤ 7 to form a discrete ylide intermediate during catalysis by PDC.