Sites of phosphorylation on pyruvate dehydrogenase from bovine kidney and heart

Abstract

The highly purified pyruvate dehydrogenase complex (EC 1.2.4.1) and uncomplexed pyruvate dehydrogenase from bovine kidney and heart mitochondria were phosphorylated and inactivated with pyruvate dehydrogenase kinase and [.gamma.-32P]ATP. Tryptic digestion of the phosphorylated pyruvate dehydrogenase yielded 3 phosphopeptides, a mono- (site 1) and a di- (sites 1 and 2) phosphorylated tetradecapeptide and a monophosphorylated nonapeptide (site 3). The amino acid sequences of the 3 phosphopeptides were established. Phosphorylation proceeded markedly faster at site 1 than at sites 2 and 3, and phosphorylation at site 1 correlated closely with inactivation of pyruvate dehydrogenase. Complete inactivation of pyruvate dehydrogenase was associated with incorporation at site 1 of 1.0-1.6 mol of phosphoryl groups/mol of enzyme. Since pyruvate dehydrogenase is a tetramer (.alpha.2.beta.2) and since phosphorylation occurs only on the .alpha. subunit, the possibility of half-site reactivity is considered.