Catalysis of acetoin formation by brewers' yeast pyruvate decarboxylase isozymes
1993
Stivers, J.T. | Washabaugh, M.W.
Catalysis of C(alpha)-proton transfer from 2-(1 hydroxyethyl)thiamin diphosphate (HETDP) by pyruvate decarboxylase isozymes (PDC; EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determining the steady-state kinetics of the reaction of [1-L]acetaldehyde (L = H, D, or T) to form acetoin and the primary kinetic isotope effects on the reaction. The PDC isozyme mixture and a4 isozyme (alpha4-PDC) have different steady-state kinetic parameters and isotope effects for acetoin formation in the presence and absence of the nonsubstrate allosteric effector pyruvamide pyruvamide activation occurs by stabilization of the acetaldehyde/PDC ternary complex. The magnitudes of primary L(V/K)-type (L = D or T) isotope effects on C(alpha)-proton transfer from alpha4-PDC-bound HETDP provide no evidence for significant breakdown of the Swain-Schaad relationship that would indicate partitioning of the putative C(alpha)-carbanion/enamine intermediate between HETDP and products. The substrate concentration dependence of the deuterium primary kinetic isotope effects provides evidence for an intrinsic isotope effect of 4.1 for C(alpha)-proton transfer from alpha4-PDC-bound HETDP. A1.10 +/- 0.02-foldl4C isotope discrimination against [ 1,2-14C] acetaldehyde in acetoin formation is inconsistent with a stepwise mechanism, in which the addition step occurs after rate-limiting formation of the C(alpha)-carbanion/enamine as a discrete enzyme-bound intermediate, and provides evidence for a concerted reaction mechanism with an important component of carbon-carbon bond formation in the transition state.
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