A simple method was developed for assessing the intramolecular coupling of active sites in the lipoate acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complexes from Escherichia coli, Bacillus stearothermophilus and ox heart and pig heart mitochondria. Samples of enzyme complex were prepared in which the pyruvate decarboxylase (E1) component was selectively and partly inhibited by treatment with increasing amounts of a transition-state analogue, thiamin thio-thiazolone pyrophosphate. The fraction of the E2 component acetylated by incubation with [2-14C] pyruvate, in the absence of CoA, was determined for each sample of partly inhibited enzyme and was found in all cases to exceed the fraction of overall complex activity remaining. This indicated the potential for transacetylation reactions among the lipoic acid residues within the E2 core. A graphic presentation of the data allowed comparison of the active-site coupling in the various enzymes, which may differ in their lipoic acid content (one or two residues per E2 chain). It is clear that active-site coupling is a general property of pyruvate dehydrogenase complexes of octahedral and icosahedral symmetries, the large numbers of subunits in each E2 core enhancing the effect.

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