Carbon Kinetic Isotope Effects on Pyruvate Decarboxylation Catalyzed by Yeast Pyruvate Decarboxylase and Models

Carbon-13 kinetic isotope effects were determined on pyruvate decarboxylation catalyzed by the enzyme yeast pyruvate decarboxylase and by thiamin, and in CHDT+ Cl- (2-(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 CO2 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 1.002 at pH 7.5 to 1.011 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.