Inhibition of the Escherichia coli pyruvate dehydrogenase complex E1 subunit and its tyrosine 177 variants by thiamin 2-thiazolone and thiamin 2-thiothiazolone diphosphates: Evidence for reversible tight-binding inhibition

Natalia Nemeria, Yan Yan, Zhen Zhang, Angela M. Brown, Palaniappa Arjunan, William Furey, John R. Guest, Frank Jordan

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Variants of the pyruvate dehydrogenase subunit (El; EC of the Escherichia coli pyruvate dehydrogenase multienzyme complex with Y177A and Y177F substitutions were created. Both variants displayed pyruvate dehydrogenase multienzyme complex activity at levels of 11% (Y177A E1) and 7% (Y177F E1) of the parental enzyme. The Km values for thiamin diphosphate (ThDP) were 1.58 μM (parental E1) and 6.65 μM (Y177A E1), whereas the Y177F E1 variant was not saturated at 200 μM. According to fluorescence studies, binding of ThDP was unaffected by the Tyr177 substitutions. The ThDP analogs thiamin 2-thiazolone diphosphate (ThTDP) and thiamin 2-thiothiazolone diphosphate (ThTTDP) behaved as tight-binding inhibitors of parental E1 (Ki = 0.003 μM for ThTDP and K i = 0.064 μM for ThTTDP) and the Y177A and Y177F variants. This analysis revealed that ThTDP and ThTTDP bound to parental E1 via a two-step mechanism, but that ThTDP bound to the Y177A variant via a one-step mechanism. Binding of ThTDP was affected and that of ThTTDP was unaffected by substitutions at Tyr177. Addition of ThDP or ThTDP to parental E1 resulted in similar CD spectral changes in the near-UV region. In contrast, binding of ThTTDP to either parental E1 or the Y177A and Y177F variants was accompanied by the appearance of a positive band at 330 nm, indicating that ThTTDP was bound in a chiral environment. In combination with x-ray structural evidence on the location of Tyr177, the kinetic and spectroscopic data suggest that Tyr177 has a role in stabilization of some transition state(s) in the reaction pathway, starting with the free enzyme and culminating with the first irreversible step (decarboxylation), as well as in reductive acetylation of the dihydrolipoamide acetyltransferase component.

Original languageEnglish (US)
Pages (from-to)45969-45978
Number of pages10
JournalJournal of Biological Chemistry
Issue number49
Publication statusPublished - Dec 7 2001


All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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