MUTAGENESIS STUDIES OF THE PHOSPHORYLATION SITES OF RECOMBINANT HUMAN PYRUVATE-DEHYDROGENASE - SITE-SPECIFIC REGULATION

Mammalian pyruvate dehydrogenase (alpha(2) beta(2)) (E(1)) is regulated by phosphorylation-dephosphorylation, catalyzed by the E(1)-kinase and the phospho-E(1)-phosphatase. Using site-directed mutagenesis of the three phosphorylation sites (sites 1, 2, and 3) on E(1) alpha, several human E(1) mutants were made with single, double, and triple mutations by changing Ser to Ala, Mutation at site 1 but not at sites 2 and/or 3 decreased E(1) specific activity and also increased K-m values for thiamin pyrophosphate and pyruvate. Sites 1, 2, and 3 in the E(1) mutants were phosphorylated either individually or in the presence of the other sites by the dihydrolipoamide acetyltransferase-protein X-E(1) kinase indicating a site-independent mechanism of phosphorylation. Phosphorylation of each site resulted in complete inactivation of the E(1). However, the rates of phosphorylation and inactivation were site-specific. Sites 1, 2, and 3 were dephosphorylated either individually or in the presence of the other sites by the phospho-E(1)-phosphatase resulting in complete reactivation of the E(1). The rates of dephosphorylation and reactivation were similar for sites 1, 2, and 3, indicating a random dephosphorylation mechanism.