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

Mammalian pyruvate dehydrogenase (alpha(2) beta(2)) (E(1)) is regulate d by phosphorylation-dephosphorylation, catalyzed by the E(1)-kinase a nd the phospho-E(1)-phosphatase. Using site-directed mutagenesis of th e three phosphorylation sites (sites 1, 2, and 3) on E(1) alpha, sever al human E(1) mutants were made with single, double, and triple mutati ons 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 fo r thiamin pyrophosphate and pyruvate. Sites 1, 2, and 3 in the E(1) mu tants were phosphorylated either individually or in the presence of th e other sites by the dihydrolipoamide acetyltransferase-protein X-E(1) kinase indicating a site-independent mechanism of phosphorylation. Ph osphorylation of each site resulted in complete inactivation of the E( 1). However, the rates of phosphorylation and inactivation were site-s pecific. Sites 1, 2, and 3 were dephosphorylated either individually o r in the presence of the other sites by the phospho-E(1)-phosphatase r esulting in complete reactivation of the E(1). The rates of dephosphor ylation and reactivation were similar for sites 1, 2, and 3, indicatin g a random dephosphorylation mechanism.