Synthetic peptide substrates for mammalian pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.
The specificities of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase were probed using synthetic peptides corresponding to the sequence around phosphorylation sites 1 and 2 on pyruvate dehydrogenase [Tyr-His-Gly-His-Ser(P1)-Met-Ser-Asp-Pro-Gly-Val-Ser(P2)-Tyr-Arg]. The dephosphotetradecapeptide containing aspartic acid at position 8 was a better substrate for the kinase than was the tetradecapeptide containing asparagine at position 8. The apparent Km and V values for the two peptides were 0.43 and 6.1 mM and 2.7 and 2.4 nmol of 32P incorporated/min/mg, respectively. Methylation of the aspartic acid residue also increased the apparent Km of the tetradecapeptide about 14-fold. These results indicate that an acidic residue on the carboxyl-terminal side of phosphorylation site 1 is an important specificity determinant for the kinase. Phosphate was incorporated only into site 1 of the synthetic peptide by the kinase. The phosphatase exhibited an apparent Km of 0.28 mM and a V of 2.3 mumol of 32P released/min/mg for the phosphorylated tetradecapeptide containing aspartic acid. Methylation of the aspartic acid residue had no significant effect on dephosphorylation. The octapeptide and phosphooctapeptide produced by cleavage of the aspartyl-prolyl bond by formic acid were poorer substrates for the kinase and phosphatase than were the tetradecapeptide and phosphotetradecapeptide, respectively. Modification of the amino terminal by acetylation or lysine addition had only a slight effect on the kinase and phosphatase activities.
D000595:Amino Acid Sequence; D001665:Binding Sites; D010455:Peptides / Q000138:chemical synthesis; D010749:Phosphoprotein Phosphatases / Q000032:analysis*; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis*; D017346:Protein Serine-Threonine Kinases; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase / Q000032:analysis*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D013379:Substrate Specificity
D010455:Peptides; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase