Secondary β-Deuterium Isotope Effects in Decarboxylation and Elimination Reactions of α-Lactylthiamin: Intrinsic Isotope Effects of Pyruvate Decarboxylase

Article abstract of DOI:10.1021/ja00284a056

The reactions of the adduct of pyruvate and thiamin, lactylthiamin (2-(lact-2-yl)thiamin), are accurate nonenzymic models for reactions of intermediates formed during catalysis by pyruvate decarboxylase.The enzymatic reaction generates lactylthiamin diphosphate from pyruvate and thiamin diphosphate. β-Deuterium isotope effects were determined for the nonenzymic reactions, and the results were related to isotope effects on the enzymic reaction. 2-(Lact-2-yl-β-d3)thiamin was prepared by condensation of methyl pyruvate-d3 with thiamin followed by hydrolysis.The isotope effect for decarboxylation of lactylthiamin in acidic solution at 25 deg C (k_H3/k_D3) is 1.09 (standard deviation (SD) 0.015) in pH 3.8, 0.5 M sodium acetate: isotope effect = 1.095 (SD 0.014) in 0.001 M HCl.The reaction was also studied using 38 percent ethanolic aqueous sodium acetate (pH 3.8 before mixing with ethanol) since the enzymic sites are less polar than water and the reaction is significantly accelerated by the cosolvent.The isotope effect is within statistical range of that for the reaction in water, 1.105 (SD 0.016), indicating that acceleration by the solvent does not change the extent of hyperconjugative stabilization of the transition state relative to the ground state.The isotope effect for the base-catalysed elimination of pyruvate from lactylthiamin was determined from kinetic studies by using multiwavelength analysis for reactions in pH 11 sodium carbonate solution.The isotope effect (k_H3/k_D3) is 1.12 (SD 0.01), which is slightly higher than the effect on decarboxylation.The isotope effets indicate that there is considerable interaction between the methyl protons of the lactyl group and the incipient electron-rich center which develops as either carbon dioxide or pyruvate is lost.The isotope effects are slightly larger than the effect reported for the catalytic rate in pyruvate decarboxylase with pyruvate-d3 as a substrate and inverse to the effects for V/K terms.This suggests that the enzymic reaction is subject to rate-limiting processes which are less isotope sensitive but that the reactions of enzyme-bound lactylthiamin diphosphate probably account for most of the observed effect on k_cat in the enzymic reaction.