Amino acid sequences around the sites of phosphorylation in the pig heart pyruvate dehydrogenase complex

Abstract

When pig heart pyruvate dehydrogenase complex was phosphorylated to completion with [.gamma.-32P]ATP by its intrinsic kinase, 3 phosphorylation sites were observed. The amino acid sequences around these sites were: sequence 1, Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg; and sequence 2, Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser(P)-Tyr-Arg. When phosphorylated to inactivation by repetitive additions of limiting quantities of [.gamma.-32P]ATP, phosphate was incorporated mainly (more than 90%) into Ser-5 of sequence 2. Phosphorylation of this site thus results in inactivation of pyruvate dehydrogenase. If Ser-5 is phosphorylated with ATP and the enzyme then incubated with [.gamma.-32P]ATP, phosphorylation of the remaining sites occurred. Ser-12 of sequence 2 is phosphorylated about twice as rapidly as Ser-6 of sequence 1. Incubation of pyruvate dehydrogenase with excess [.gamma.-32P]ATP with termination of phosphorylation at .apprx. 50% complete inactivation showed that Ser-5 of sequence 2 was phosphorylated most rapidly, but also that Ser-12 of sequence 2 was significantly (15% of total) phosphorylated. Ser-6 of sequence 1 contained about 1% total P. These results suggest that addition of limiting amounts of ATP primarily produces phosphorylation of Ser-5 of sequence 2 (inactivating site). This also occurs during incubation with excess ATP before complete inactivation occurs, but a greater occupancy of other sites also occurs during this treatment.