| Abstract: | The dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex catalyzes a reversible reaction between acetyl-CoA and dihydrolipoamide that results in the formation of S-acetyldihydrolipoamide. We have used 13C nuclear magnetic resonance to investigate this reaction using exogenous forms of dihydrolipoamide in place of the protein-bound substrate. With substrate levels of dihydrolipoamide and enzymatically generated 1-13C]acetyl-CoA, both 6-S-1-13C]acetyl- and 8-S-1-13C]acetyldihydrolipoamide were formed in the transacetylation reaction and both species participated in the reverse reaction to yield 1-13C]acetyl-CoA and free dihydrolipoamide. The 8-S-acetyl derivative was the principal product. It is suggested that acetylation of both the 6- and 8-thiols of dihydrolipoamide results as a consequence of intramolecular migration following acetylation at a single site. After longer periods of reaction, some 6,8-S,S-1-13C]diacetyldihydrolipoamide also accumulated. We have also found that 1-13C]acetyl-CoA reacts slowly with dihydrolipoamide in a nonenzymatic reaction to yield the two monoacetylated and some diacetylated derivative. In the reverse reaction catalyzed by the dihydrolipoyl transacetylase, it was clear that monoacetyl derivatives were depleted much more rapidly than the diacetyl derivatives, although we could not quantitate the change in the low concentration of the diacetyl derivative. |
