Detection and time course of formation of major thiamin diphosphate-bound covalent intermediates derived from a chromophoric substrate analogue on benzoylformate decarboxylase.

ABSTRACT: The mechanism of the enzyme benzoylformate decarboxylase (BFDC), which carries out a typical thiamin diphosphate (ThDP)-dependent nonoxidative decarboxylation reaction, was studied with the chromophoric alternate substrate (E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid (3-PKB). Addition of 3-PKB resulted in the appearance of two transient intermediates formed consecutively, the first one to be formed a predecarboxylation ThDP-bound intermediate with lambda(max) at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with lambda(max) at 437 nm. The time course of formation/depletion of the PKB-ThDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB-ThDP covalent adduct. When the product of decarboxyla

SUBMITTER: Chakraborty S 

PROVIDER: S-EPMC2788921 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature