Role of isoleucine 415 as a conformational pivot in yeast pyruvate decarboxylase

Frank Jordan, Fusheng Guo, Ueqi Zhang, Ara Kahyaoglu, Ramy Farid

Research output: Contribution to journalArticlepeer-review

Abstract

In each of the thiamin diphosphate-dependent enzymes with published crystal structures, there is found a hydrophobic amino acid side chain wedged in the crevice of the aeV coenzyme conformation (the relative disposition of the 4:amino-2'-methylpyrimidine and thiazolium rings with respect to the bridging méthylène group). It was suggested by the authors' before that this conformation would bring the 4'-amino group within close proximity of the reactive C2 thiazolium atom, so that the imino tautomer of the 4'-amino group could accept a proton to generate the required ylide/carbene (conjugate base of the thiazolium ring). Isoleucine 415, the side chain found in this crevice of pyruvate decarboxylase from Saccharomyces cerevisiae has been converted to a variety of uncharged side chains. In the series of Ile, Val, Thr, the kcat decreases with decreasing size of the side chain, and more surprisingly, the kcat-pH profile is shifted to more alkaline values with decreasing size of the side chain. Ile415 therefore also affects the disposition of the groups responsible for the pH pro files. On the other hand, the Hill coefficient appears to be little affected by the substitutions at this position, indicating that this residue does not participate in the substrate activation pathway. Computational studies showed that the 1415 side chain is not required for the seV conformational preference, but that there likely is an entropie, as well as a hydrophobic contribution of 1415 to the catalysis. Supported by NIH- GM 50380, the Rutgers Busch Fund and Hoffmann LaRoche Diagnostics Division.

Original languageEnglish (US)
Pages (from-to)A1331
JournalFASEB Journal
Volume12
Issue number8
StatePublished - 1998

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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