Carbon kinetic isotope effects on pyruvate decarboxylation catalyzed by yeast pyruvate decarboxylase and models

Abstract

Carbon-1 3 kinetic isotope effects were determined on pyruvate decarboxylation catalyzed by the enzyme yeast pyruvate decarboxylase and by thiamin, and in CHDT+Cl-( 2 4 1-carboxy-1 -hydroxyethyl)-3,4-dimethylthiazolium chloride). The CHDT+Cl-gave an effect of 1.051 corresponding to the maximum isotope effect anticipated for COz loss. Thiamin-catalyzed decarboxylation gave a pH-independent inverse isotope effect of 0.992 indicating that in that model decomposition of the covalent adduct formed between thiamin and pyruvate to reactants has a higher activation energy than the subsequent decarboxylation step. The enzymatic isotope effect was found to be normal varying from l .002 at pH 7.5 to 1.01 l at pH 5.0. At pH 5.00 the isotope effect was found to be temperature independent. The results were interpreted to mean that in the pH range employed decarboxylation is faster than the decomposition of the enzyme-bound thiamin-pyruvate covalent complex. A model is presented to account for the observed pH dependence of the enzymatic kinetic isotope effect.Yeast pyruvate decarboxylase (EC 4.1.1.1) (PDCase) catalyzes the decarboxylation of pyruvate to acetaldehyde with the assistance of thiamin pyrophosphate and Mg(1I) as cofactors.* Numerous studies have shown that pyruvate and the coenzyme form a covalent complex. In fact, 2-(cr-hydroxy)ethylthiamin pyrophosphate (the adduct of acetaldehyde and the coenzyme) can be isolated from the enzymic r e a~t i o n .~ Scheme I accounts for the known facts.Carbon kinetic isotope effects on the release of C 0 2 can elucidate whether the decarboxylation is the slow, rate-controlling step in the series of steps culminating with C02 release (assuming this last step to be essentially irreversible). While few such experiments have been reported (Seltzer et al. on oxaloacetate decarbo~ylose,~ O'Leary on isocitrate dehydrog e n a~e ,~ acetoacetate decarboxylase,6 and glutamate decar-0002-1863/78/1500-2872$01 .OO/O Scheme I 6H / \ CH, OH boxylase,' and Cleland on malic enzyme8), they provide information not readily accessible by any other technique.

Fractionation of carbon (13C/12C) isotopes in glycine decarboxylase reaction

“…E-mail: ivlev@ivlev.msk.ru carboxylase reaction in vitro. We have anticipated that CO2 evolved in the reaction should be enriched in 12C compared to the initial substrate as was observed before in the other enzymatic decarboxylation reactions [6][7][8][9]. However, we have found that the carbon isotope effect in the above reaction was in most cases of the opposite sign and CO2 was enriched in 13C.…”

Stable isotope discrimination during soil denitrification: Production and consumption of nitrous oxide

“…Rather, we suspect that the shift in the direction of the fractionation likely involves other processes. First, there may be an enzyme system capable of consuming N 2 O that causes inverse fractionation, a phenomenon that has been observed for other enzyme systems [ Alkema et al , 2003; Jordan et al , 1978]. As described above, the enzyme characteristics of nitrous oxide reductase have been characterized from only a few denitrifying bacteria.…”

Kinetics and mechanism of benzoylformate decarboxylase using carbon-13 and solvent deuterium isotope effects on benzoylformate and benzoylformate analogs

“…13C isotope effects for an analogous reaction catalyzed by pyruvate decarboxylase from yeast have been reported. O'Leary (1976) determined that 13( V/K) for pyruvate was 1.0083 at pH 6.8 and 25 "C. Jordan et al (1978) found that 13( V/K) for pyruvate varied with pH from 1.011 at pH 5 to 1.002 at pH 7.5 and 30 "C. Interestingly, Melzer and Schmidt (1987) determined that the 13C isotope effect at C-1 of pyruvate was 1.0093 (Escherichia coli, pH 7.6 and 36 "C) and 1.024 (yeast, pH 7.8 and 36 "C) for the overall reaction catalyzed by the pyruvate dehydrogenase multienzyme complex.…”