Evidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment

TitleEvidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment
Publication TypeJournal Article
Year of Publication2005
AuthorsZhang, S, Zhou L, Nemeria N, Yan Y, Zhang Z, Zou Y, Jordan F
JournalBiochemistry
Volume44
Pagination2237-2243
Date PublishedFeb
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=000227075200001
KeywordsCATALYSIS, COENZYME, DECARBOXYLATIONS, DEHYDROGENASE MULTIENZYME COMPLEX, DEPENDENT, ESCHERICHIA-COLI, INTERMEDIATE, KINETICS, LIPOYL DOMAINS, PYRUVATE, REDUCTIVE ACETYLATION
Abstract

The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment that helps to stabilize unstable zwitterionic/dipolar intermediates (including the enamine/C2alpha-carbanion present on all such enzymes) was tested experimentally using the intermediate C2alpha-hydroxyethylthiamin diphosphate (HEThDP) with the Escherichia coli pyruvate dehydrogenase complex and its E1 subunit (PDHc-E1). Using pre-steadystate and steady-state methods, it was shown that HEThDP is a substrate for this enzyme after ionization of the C2alpha-H bond. An experiment was then carried out to measure the PDHc-E1 catalyzed pre-steadystate rate constant for the D --> H exchange from the C2alpha position of HEThDP-d(4), as an indicator of the formation of the enamine. Importantly, the enzyme accelerates the rate of ionization of this bond by a factor of 10(7), corresponding to a 10 kcal/mol stabilization of the enamine intermediate by the enzyme. This finding is likely a general feature of thiamin diphosphate enzymes.

URLhttp://apps.isiknowledge.com/InboundService.do?Func=Frame&product=WOS&action=retrieve&SrcApp=EndNote&Init=Yes&SrcAuth=ResearchSoft&mode=FullRecord&UT=000227075200001
Alternate JournalBiochemistry