Direct observation of the kinetic fate of a thiamin diphosphate bound enamine intermediate on Brewers' yeast pyruvate decarboxylase. Kinetic and regiospecific consequences of allosteric activation

Article abstract of DOI:10.1021/ja00015a044

The interaction of (E)-2-oxo-4-p-tolyl-3-butenoic acid with brewer's yeast pyruvate decarboxylase was examined by kinetic and absorption spectroscopic means. The compound undergoes catalytic turnover, leading to a thiamin diphosphate bound enamine intermediate that can be protonated at both allylic positions leading to p-methylcinnamaldehyde and p-methyldihydrocinnamic acid in a ratio of 1:3 in the absence of and 3:2 in the presence of the allosteric activator pyruvamide. The compound is also a weak but irreversible inactivator of the enzyme, and according to colorimetric titration of the enzyme prior to and subsequent to inactivation, it reacts with a Cys side chain with a 1:1 stoichiometry; i.e., one Cys/subunit is being modified. The data are consistent with partitioning of the 2-(p-methyldihydrocinnamoyl)thiamin diphosphate between nonenzymic hydrolysis to the free p-methyldihydrocinnamic acid and transfer to a Cys on the enzyme forming a p-methyldihydrocinnamoyl thiol ester. The latter at the pH used is quite stable, and hence inactivates the enzyme. An enzyme-bound enamine intermediate can be detected at 440 nm, and its rates of formation and disappearance can be conveniently monitored. A thiazolium model for a precursor to such an enamine, 2-[γ-[tetrahydro-2H-pyran-2-yl)oxy]-(E)-cinnamyl]-3,4,5- trimethylthiazolium ion, when treated with (TMS)₂NNa in Me₂SO gave an absorbance with identical λ_max, confirming the assignment of the absorbance observed from such conjugated 2-keto acids to the thiamin-bound enamine structure. Finally, the allosteric activator pyruvamide was found to enhance the rate of enamine formation by as much as 50-fold, both the rates of formation and conversion to product of the enamine being affected by the allosteric regulation. On the basis of data provided, (E)-2-oxo-4-p-tolyl-3-butenoic acid is proposed as a convenient active-site titrant for pyruvate decarboxylase.