The 1 ',4 '-iminopyrimidine tautomer of thiamin diphosphate is poised for catalysis in asymmetric active centers on enzymes

TitleThe 1 ',4 '-iminopyrimidine tautomer of thiamin diphosphate is poised for catalysis in asymmetric active centers on enzymes
Publication TypeJournal Article
Year of Publication2007
AuthorsNemeria, N, Chakraborty S, Baykal A, Korotchkina LG, Patel MS, Jordan F
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Pagination78-82
Date PublishedJan
Type of ArticleArticle
ISBN Number0027-8424
Accession Numberhttp://apps.isiknowledge.com/InboundService.do?Func=Frame&product=WOS&action=retrieve&SrcApp=EndNote&Init=Yes&SrcAuth=ResearchSoft&mode=FullRecord&UT=000243456300016
Keywords1 ',4 '-iminothiamin diphospate, 2-oxoacid decarboxylases, ACID-BASE GROUPS, active site, ANGSTROM RESOLUTION, asymmetry, BINDING INHIBITION, DECARBOXYLASE, DEHYDROGENASE MULTIENZYME COMPLEX, DEPENDENT ENZYMES, E1 SUBUNIT, ESCHERICHIA-COLI, HUMAN PYRUVATE-DEHYDROGENASE, REACTION INTERMEDIATE
Abstract

Thiamin diphosphate, a key coenzyme in sugar metabolism, is comprised of the thiazolium and 4'-aminopyrimidine aromatic rings, but only recently has participation of the 4'-aminopyrimidine moiety in catalysis gained wider acceptance. We report the use of electronic spectroscopy to identify the various tautomeric forms of the 4'-aminopyrimidine ring on four thiamin diphosphate enzymes, all of which decarboxylate pyruvate: the Ell component of human pyruvate dehydrogenase complex, the Ell subunit of Escherichia coli pyruvate dehydrogenase complex, yeast pyruvate decarboxylase, and pyruvate oxidase from Lactobacillus plantarum. It is shown that, according to circular dichroism spectroscopy, both the 1',4'-iminopyrimidine and the 4'-aminopyrimidine tautomers coexist on the Ell component of human pyruvate dehydrogenase complex and pyruvate oxidase. Because both tautomers are seen simultaneously, these two enzymes provide excellent evidence for nonidentical active centers (asymmetry) in solution in these multimeric enzymes. Asymmetry of active centers can also be induced upon addition of acetylphosphinate, an excellent electrostatic pyruvate mimic, which participates in an enzyme-catalyzed addition to form a stable adduct, resembling the common predecarboxylation thiamin-bound intermediate, which exists in its 1',4'-iminopyrimidine form. The identification of the 1',4'-iminopyrimidine tautomer on four enzymes is almost certainly applicable to all thiamin diphosphate enzymes: this tautomer is the intramolecular trigger to generate the reactive ylide/carbene at the thiazolium C2 position in the first fundamental step of thiamin catalysis.

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Alternate JournalProc. Natl. Acad. Sci. U. S. A.