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

Variants of the pyruvate dehydrogenase subunit (El; EC 1.2.4.1) of the Esch erichia coli pyruvate dehydrogenase multienzyme complex with Y177A and Y177 F substitutions were created. Both variants displayed pyruvate dehydrogenas e multienzyme complex activity at levels of 11% (Y177A El) and 7% (Y177F El ) of the parental enzyme. The K-m values for thiamin diphosphate (ThDP) wer e 1.58 mum (parental El) and 6.65 mum (Y177A EI), whereas the Y177F El vari ant was not saturated at 200 mum. According to fluorescence studies, bindin g of ThDP was unaffected by the Tyr(117) substitutions. The ThDP analogs th iamin 2-thiazolone diphosphate (ThTDP) and thiamin 2-thiothiazolone diphosp hate (ThTTDP) behaved as tight-binding inhibitors of parental El (K-i = 0.0 03 mum for ThTDP and K-i = 0.064 mum for ThTTDP) and the Y177A and Y177F va riants. This analysis revealed that ThTDP and ThTTDP bound to parental El v ia a two-step mechanism, but that ThTDP bound to the Y177A variant via a on e-step mechanism. Binding of ThTDP was affected and that of ThTTDP was unaf fected by substitutions at Tyr(177). Addition of ThDP or ThTDP to parental El resulted in similar CD spectral changes in the near-UV region. In contra st, binding of ThTTDP to either parental El 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 s tructural evidence on the location of Tyr(177), the kinetic and spectroscop ic data suggest that Tyr(177) has a role in stabilization of some transitio n state(s) in the reaction pathway, starting with the free enzyme and culmi nating with the first irreversible step (decarboxylation), as well as in re ductive acetylation of the dihydrolipoamide acetyltransferase component.