Mechanism of benzaldehyde lyase studied via thiamin diphosphate-bound intermediates and kinetic isotope effects

TitleMechanism of benzaldehyde lyase studied via thiamin diphosphate-bound intermediates and kinetic isotope effects
Publication TypeJournal Article
Year of Publication2008
AuthorsChakraborty, S, Nemeria N, Yep A, McLeish MJ, Kenyon GL, Jordan F
JournalBiochemistry
Volume47
Pagination3800-3809
Date PublishedMar
Type of ArticleArticle
ISBN Number0006-2960
Accession Numberhttp://apps.isiknowledge.com/InboundService.do?Func=Frame&product=WOS&action=retrieve&SrcApp=EndNote&Init=Yes&SrcAuth=ResearchSoft&mode=FullRecord&UT=000254127900023
Keywords1',4'-IMINOPYRIMIDINE TAUTOMER, ACTIVE-SITE, BENZOYLFORMATE DECARBOXYLASE, CATALYSIS, CRYSTALLOGRAPHIC SNAPSHOTS, DECARBOXYLATIONS, DEHYDROGENASE COMPLEX, DEPENDENT, ENAMINE INTERMEDIATE, PSEUDOMONAS-FLUORESCENS, YEAST PYRUVATE DECARBOXYLASE
Abstract

Direct spectroscopic observation of thiamin diphosphate-bound intermediates was achieved on the enzyme benzaldehyde lyase, which carries out reversible and highly enantiospecific conversion of (R)-benzoin to benzaldehyde. The key enamine intermediate could be observed at lambda(max) 393 nm in the benzoin breakdown direction and in the decarboxylase reaction starting with benzoylformate. With benzaldehyde as substrate, no intermediates could be detected, only formation of benzoin at 314 nm. To probe the rate-limiting step in the direction of (R)-benzoin synthesis, the H-1/H-2 kinetic isotope effect was determined for benzaldehyde labeled at the aldehyde position and found to be small (1.14 +/- 0.03), indicating that ionization of the C2 alpha H from C2 alpha-hydroxybenzylthiamin diphosphate is not rate limiting. Use of the alternate substrates benzoylformic and phenylpyruvic acids (motivated by the observation that While a carboligase, benzaldehyde lyase could also catalyze the slow decarboxylation of 2-oxo acids) enabled the observation of the substrate-thiamin covalent intermediate via the 1',4'-iminopyrimidine tautomer, characteristic of all intermediates with a tetrahedral C2 substituent on ThDP. The reaction of benzaldehyde lyase with the chromophoric substrate analogue (E)-2-6xo-4(pyridin-3-yl)-3-butenoic acid and its decarboxylated product (E)-3-(pyridine-3-yl)acrylaldehyde enabled the detection of covalent adducts with both. Neither adduct underwent further reaction. An important finding of the studies is that all thiamin-related intermediates are in a chiral environment on benzaldehyde lyase as reflected by their circular dichroism signatures.

URLhttp://apps.isiknowledge.com/InboundService.do?Func=Frame&product=WOS&action=retrieve&SrcApp=EndNote&Init=Yes&SrcAuth=ResearchSoft&mode=FullRecord&UT=000254127900023
Alternate JournalBiochemistry