Sites of phosphorylation on pyruvate dehydrogenase from bovine kidney and heart

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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.