CATALYSIS OF ACETOIN FORMATION BY BREWERS-YEAST PYRUVATE DECARBOXYLASE ISOZYMES

Catalysis of C(alpha)-proton transfer from 2-(1-hydroxyethyl)thiamin d iphosphate (HETDP) by pyruvate decarboxylase isozymes (PDC; EC 4.1.1.1 ) from Saccharomyces carlsbergensis was investigated by determining th e 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 alpha4 isozyme (alpha4-PDC) hav e different steady-state kinetic parameters and isotope effects for ac etoin formation in the presence and absence of the nonsubstrate allost eric effector pyruvamide: pyruvamide activation occurs by stabilizatio n 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 fr om alpha4-PDC-bound HETDP provide no evidence for significant breakdow n of the Swain-Schaad relationship that would indicate partitioning of the putative C(alpha)-carbanion/enamine intermediate between HETDP an d products. The substrate concentration dependence of the deuterium pr imary kinetic isotope effects provides evidence for an intrinsic isoto pe effect of 4.1 for C(alpha)-proton transfer from alpha4-PDC-bound HE TDP. A1.10 +/- 0.02-fold C-14 isotope discrimination against [1,2-C-14 ] acetaldehyde in acetoin formation is inconsistent with a stepwise me chanism, in which the addition step occurs after rate-limiting formati on of the C(alpha)-carbanion/enamine as a discrete enzyme-bound interm ediate, and provides evidence for a concerted reaction mechanism with an important component of carbon-carbon bond formation in the transiti on state.