A kinetic study of dihydrolipoyl transacetylase from bovine kidney

Abstract

The mammalian pyruvate dehydrogenase complex contains a core, consisting of dihydrolipoyl transacetylase, to which pyruvate dehydrogenase and dihydrolipoyl dehydrogenase are joined. This report describes studies on the kinetic mechanism of the transacetylase-catalyzed reaction between [1-14C]acetyl-CoA and dihydrolipoamide. This reaction appears to be a model of the physiological reaction, in which the acetyl group is transferred from the S-acetyldihydrolipoyl moiety, bound covalently to the transacetylase, to CoA. The model reaction is not affected by pyruvate dehydrogenase or dihydrolipoyl dehydrogenase, their substrates and products, or by removal of the covalently bound lipoyl moiety. These findings, together with the results of initial velocity, product inhibition, and dead-end inhibition studies, indicate that the model reaction and, apparently, the physiological reaction as well, proceeds via the Random Bi Bi (rapid equilibrium) mechanism. It appears that at the catalytic center of the transacetylase there are two adjacent sites, one that binds CoA and acetyl-CoA and another that binds dihydrolipoamide and S-acetyldihydrolipoamide (or the corresponding forms of the covalently bound lipoyl moiety.