NMR analysis of covalent intermediates in thiamin diphosphate enzymes

TitleNMR analysis of covalent intermediates in thiamin diphosphate enzymes
Publication TypeJournal Article
Year of Publication2003
AuthorsTittmann, K, Golbik R, Uhlemann K, Khailova L, Schneider G, Patel M, Jordan F, Chipman DM, Duggleby RG, Hubner G
JournalBiochemistry
Volume42
Pagination7885-7891
Date PublishedJul
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=000183957900003
KeywordsACID-BASE, ACTIVE-SITE, ANGSTROM RESOLUTION, CATALYSIS, CRYSTAL-STRUCTURE, DEPENDENT ENZYME, GROUPS, SACCHAROMYCES-CEREVISIAE, SITE-DIRECTED MUTAGENESIS, YEAST PYRUVATE DECARBOXYLASE, ZYMOMONAS-MOBILIS
Abstract

Enzymic catalysis proceeds via intermediates formed in the course of substrate conversion. Here, we directly detect key intermediates in thiamin diphosphate (ThDP)-dependent enzymes during catalysis using H-1 NMR spectroscopy. The quantitative analysis of the relative intermediate concentrations allows the determination of the microscopic rate constants of individual catalytic steps. As demonstrated for pyruvate decarboxylase (PDC), this method, in combination with site-directed mutagenesis, enables the assignment of individual side chains to single steps in catalysis. In PDC, two independent proton relay systems and the stereochemical control of the enzymic environment account for proficient catalysis proceeding via intermediates at carbon 2 of the enzyme-bound cofactor. The application of this method to other ThDP-dependent enzymes provides insight into their specific chemical pathways.

URLhttp://apps.isiknowledge.com/InboundService.do?Func=Frame&product=WOS&action=retrieve&SrcApp=EndNote&Init=Yes&SrcAuth=ResearchSoft&mode=FullRecord&UT=000183957900003
Alternate JournalBiochemistry