Tetrahedral intermediates in thiamin diphosphate-dependent decarboxylations exist as a 1 ',4 '-imino tautomeric form of the coenzyme, unlike the Michaelis complex or the free coenzyme

TitleTetrahedral intermediates in thiamin diphosphate-dependent decarboxylations exist as a 1 ',4 '-imino tautomeric form of the coenzyme, unlike the Michaelis complex or the free coenzyme
Publication TypeJournal Article
Year of Publication2004
AuthorsNemeria, N, Baykal A, Joseph E, Zhang S, Yan Y, Furey W, Jordan F
JournalBiochemistry
Volume43
Pagination6565-6575
Date PublishedJun
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=000221658000021
KeywordsACID-BASE GROUPS, ANGSTROM RESOLUTION, DEHYDROGENASE MULTIENZYME COMPLEX, E1 SUBUNIT, ENZYME, ESCHERICHIA-COLI, inhibition, PHOSPHONATE, TRANSKETOLASE, YEAST PYRUVATE DECARBOXYLASE
Abstract

Two circular dichroism signals observed on thiamin diphosphate (ThDP)-dependent enzymes, a positive band in the 300-305 nm range and a negative one in the 320-330 nm range, were investigated on yeast pyruvate decarboxylase (YPDC) and on the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1). Addition of the tetrahedral ThDP-acetaldehyde adduct, 2-alpha-hydroxyethylThDP, to PDHc-E1 generates the positive band at 300 nm, consistent with the formation of the 1',4'-iminopyrimidine tautomer, as also demonstrated for phosphonolactylthiamin diphosphate, a stable analogue of the tetrahedral ThDP-pyruvate adduct 2-alpha-lactylThDP (Jordan, F. et al. (2003) J. Am. Chem. Soc. 125, 12732-12738). Therefore, we suggest that all tetrahedral ThDP-bound covalent complexes will also prefer this tautomer, and that the 4'-aminopyrimidine of ThDP participates in multiple steps of acid-base catalysis on ThDP enzymes. Studies with YPDC and PDHc-E1, and their active center variants, in conjunction with chemical models, enabled assignment of the negative band at 330 nm to a charge-transfer transition between the 4'-aminopyrimidine tautomer (presumed electron donor) and the thiazolium ring (presumed electron acceptor) of ThDP, with no significant contributions from any amino acid side chain of the proteins. However, in both YPDC and PDHc-E1, the presence of substrate or substrate surrogate was required to enable detection, suggesting that the band at 320-330 nm be used as a reporter for the Michaelis complex, involving the amino tautomer, on both enzymes. As the positive band near 300 nm reports on the 1',4'-imino tautomer of ThDP, methods are now available for kinetic monitoring of both tautomeric forms.

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Alternate JournalBiochemistry