Effect of Substitutions in the Thiamin Diphosphate-Magnesium Fold on the Activation of the Pyruvate Dehydrogenase Complex from Escherichia coli by Cofactors and Substrate

Abstract

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.

The E2 Domain of OdhA of Corynebacterium glutamicum Has Succinyltransferase Activity Dependent on Lipoyl Residues of the Acetyltransferase AceF

“…and for AceE and pyruvate a K m value of 0.066 Ϯ 0.06 mM were derived. The value for AceE is close to that known for the E1 reaction of the E. coli PDH, for which the K m is 0.110 mM (16), or that of human E1 protein, for which it is 0.026 mM (38). Mutations in the lipoyl domains.…”

Glutamate 636 of the Escherichia coli Pyruvate Dehydrogenase-E1 Participates in Active Center Communication and Behaves as an Engineered Acetolactate Synthase with Unusual Stereoselectivity

“…4A for the E636Q variant). In contrast, whereas the 3-lipoyl domain and 1-lipoyl domain PDHcs and the E1 subunit resolved from the former all showed a lag phase on ThDP binding, with each the lag phase disappeared at Ͼ25 M concentration of ThDP (15). The presence of this lag phase implies a slow isomerization step subsequent to binding of ThDP.…”

Novel Binding Motif and New Flexibility Revealed by Structural Analyses of a Pyruvate Dehydrogenase-Dihydrolipoyl Acetyltransferase Subcomplex from the Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex

“…1). The 1-lip E2p displays similar kinetic behavior as the 3-lip E2p according to the NADH (overall complex) assay, indicating the LD h is functionally competent (48).…”