Effect of substitutions in the thiamin diphosphate-magnesium fold on the activation of the pyruvate dehydrogenase complex from Escherichia coli by cofactors and substrate.

The homotropic regulation of the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) by its coenzyme thiamin diphosphate and its substrate pyruvate was re-examined with complexes containing three and one lipoyl domains per E2 chain, and several variants of the latter, containing substitutions in the putative thiamin diphosphate fold of E1 (G231A, G231S, C259S, C259N, and N258Q). It was found that all of the E1 variants had significantly reduced specific activities, as reported elsewhere (Russell, G. C., Machado, R. S., and Guest, J. R. (1992) Biochem. J. 287, 611-619). In addition, extensive kinetic studies were performed in an attempt to determine the effects of the amino acid substitutions on the Hill coefficients with respect to thiamin diphosphate and pyruvate. All but one of the variants were incapable of being saturated with thiamin diphosphate, even at concentrations > 5 mM. Most importantly, the striking activation lag phase lasting for many seconds in the parental complexes containing three and one lipoyl domains per E2 chain was totally eliminated in the variants. Furthermore, activation by the coenzyme was localized to the E1 subunit, because resolved E1 exhibits virtually the same behavior during the activation lag phase as does the complex. In the parental complexes two distinct lag phases could be resolved, the duration of both decreases with increasing ThDP concentration. A mechanism that is consistent with all of the kinetic data on the parental complexes involves rapid equilibration of the first ThDP with the E1 dimer, followed by a slow conformational equilibration, that in turn is followed by slow addition of the second ThDP to form the fully activated dimer. When the diphosphate site is badly impaired, the binding affinity is very much reduced, this perhaps eliminates the slow step leading to the activated dimer form of the E1.