Reductive acetylation of tandemly repeated lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli is random order.

In vitro deletion and site-directed mutagenesis of the aceF gene of Escherichia coli was used to generate dihydrolipoamide acetyltransferase (E2p) polypeptide chains containing various permutations and combinations of functional and non-functional lipoyl domains. A lipoyl domain was rendered non-functional by converting the lipoylatable lysine residue to glutamine. Two- and three-lipoyl domain E2p chains, with lipoyl-lysine (Lys244) substituted by glutamine in the innermost lipoyl domains (designated +/- and +/+/-, respectively), and similar chains with lipoyl-lysine (Lys143) substituted by glutamine in the outer lipoyl domains (designated -/+ and -/-/+), were constructed. In all instances, pyruvate dehydrogenase complexes were assembled in vivo around E2p cores composed of the modified peptide chains. All the complexes were essentially fully active in catalysis, although the complex containing the -/-/+ version of the E2p polypeptide chain showed a 50% reduction in specific catalytic activity. Similarly, active-site coupling in the complexes containing the +/-, +/+/- and -/+ constructions of the E2p chains was not significantly different from that achieved by the wild-type complex. However, active-site coupling in the complex containing the -/-/+ version of the E2p chain was slightly impaired, consistent with the reduced overall complex activity. These results indicate that during oxidative decarboxylation there is no mandatory order of reductive acetylation of repeated lipoyl domains within E2p polypeptide chains, and strongly suggest that the three tandemly repeated lipoyl domains in the wild-type E2p chain function independently in the pyruvate dehydrogenase complex.