Elucidation of the chemistry of enzyme-bound thiamin diphosphate prior to substrate binding: Defining internal equilibria among tautomeric and ionization states

TitleElucidation of the chemistry of enzyme-bound thiamin diphosphate prior to substrate binding: Defining internal equilibria among tautomeric and ionization states
Publication TypeJournal Article
Year of Publication2007
AuthorsNemeria, N, Korotchkina L, McLeish MJ, Kenyon GL, Patel MS, Jordan F
JournalBiochemistry
Volume46
Pagination10739-10744
Date PublishedSep
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=000249433100034
Keywords1',4'-IMINOPYRIMIDINE TAUTOMER, ACID-BASE, BENZALDEHYDE LYASE, BENZOYLFORMATE DECARBOXYLASE, CATALYSIS, COENZYME, HUMAN PYRUVATE-DEHYDROGENASE, MECHANISM, PYROPHOSPHATE, SITE
Abstract

Both solution and crystallographic studies suggest that the 4'-aminopyrimidine ring of the thiamin diphosphate coenzyme participates in catalysis, likely as an intramolecular general acid-base catalyst via the unusual 1',4'-iminopyrimidine tautomer. It is indeed uncommon for a coenzyme to be identified in its rare tautomeric form on its reaction pathways, yet this has been possible with thiamin diphosphate, in some cases even in the absence of substrate [Nemeria, N., Chakraborty, S., Baykal, A., Korotchkina, L., Patel, M. S., and Jordan, F. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 78-82.]. The ability to detect both the aminopyrimidine and iminopyrimidine tautomeric forms of thiamin diphosphate on enzymes has enabled us to assign the predominant tautomeric form present in individual intermediates on the pathway. Herein, we report the pH dependence of these tautomeric forms providing the first data for the internal thermodynamic equilibria on thiamin diphosphate enzymes for. the various ionization and tautomeric forms of this coenzyme on four enzymes: benzaldehyde lyase, benzoylformate decarboxylase, pyruvate oxidase, and the El component of the human pyruvate dehydrogenase multienzyme complex. Evidence is provided for an important function of the enzyme environment in altering both the ionization and tautomeric equilibria on the coenzyme even prior to addition of substrate. The pK(a) for the 4'-aminopyrinfidinium moiety coincides with the pH for optimum activity thereby ensuring that all ionization states and tautomeric states are accessible during the catalytic cycle. The dramatic influence of the protein on the internal equilibria also points to conditions under which the long-elusive ylide intermediate could be stabilized.

URLhttp://apps.isiknowledge.com/InboundService.do?Func=Frame&product=WOS&action=retrieve&SrcApp=EndNote&Init=Yes&SrcAuth=ResearchSoft&mode=FullRecord&UT=000249433100034
Alternate JournalBiochemistry