Catalysis of acetoin formation by brewers' yeast pyruvate decarboxylase isozymes.
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 alpha 4 isozyme (alpha 4-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 alpha 4-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 alpha 4-PDC-bound HETDP. A 1.10 +/- 0.02-fold 14C 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.
Stivers
J T
JT
Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205-2179.
Washabaugh
M W
MW
D000079:Acetaldehyde; D000093:Acetoin / Q000378:metabolism*; D000494:Allosteric Regulation; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011767:Pyruvate Decarboxylase / Q000378:metabolism*; D011773:Pyruvates / Q000494:pharmacology; D012440:Saccharomyces / Q000201:enzymology*
D007527:Isoenzymes; D011773:Pyruvates; D000093:Acetoin; D011767:Pyruvate Decarboxylase; D000079:Acetaldehyde; C009144:pyruvamide