Detection and Time Course of Formation of Major Thiamin Diphosphate-Bound Covalent Intermediates Derived from a Chromophoric Substrate Analogue on Benzoylformate Decarboxylase&#x2020

TitleDetection and Time Course of Formation of Major Thiamin Diphosphate-Bound Covalent Intermediates Derived from a Chromophoric Substrate Analogue on Benzoylformate Decarboxylase&#x2020
Publication TypeJournal Article
Year of PublicationSubmitted
AuthorsChakraborty, S, Nemeria NS, Balakrishnan A, Brandt GS, Kneen MM, Yep A, McLeish MJ, Kenyon GL, Petsko GA, Ringe D, Jordan F
JournalBiochemistry
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 位max at 477 nm, and the second one corresponding to the first postdecarboxylation intermediate the enamine with位max at 437 nm. The time course of formation/depletion of the PKB鈭扵hDP covalent complex and of the enamine showed that decarboxylation was slower than formation of the PKB鈭扵hDP covalent adduct. When the product of decarboxylation 3-(pyridin-3-yl)acrylaldehyde (PAA) was added to BFDC, again an absorbance with 位max at 473 nm was formed, corresponding to the tetrahedral adduct of PAA with ThDP. Addition of well-formed crystals of BFDC to a solution of PAA resulted in a high resolution (1.34 脜) structure of the BFDC-bound adduct of ThDP with PAA confirming the tetrahedral nature at the C2伪 atom, rather than of the enamine, and supporting the assignment of the 位max at 473 nm to the PAA鈭扵hDP adduct. The structure of the PAA鈭扵hDP covalent complex is the first example of a product鈭扵hDP adduct on BFDC. Similar studies with 3-PKB indicated that decarboxylation had taken place. Evidence was also obtained for the slow formation of the enamine intermediate when BFDC was incubated with benzaldehyde, the product of the decarboxylation reaction thus confirming its presence on the reaction pathway.

URLhttp://pubs.acs.org/doi/abs/10.1021/bi801810h