Mutagenesis Studies of the Phosphorylation Sites of Recombinant Human Pyruvate Dehydrogenase. SITE-SPECIFIC REGULATION

Abstract

Mammalian pyruvate dehydrogenase (alpha 2 beta 2) (E1) is regulated by phosphorylation-dephosphorylation, catalyzed by the E1-kinase and the phospho-E1-phosphatase. Using site-directed mutagenesis of the three phosphorylation sites (sites 1, 2, and 3) on E1 alpha, several human E1 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 E1 specific activity and also increased Km values for thiamin pyrophosphate and pyruvate. Sites 1, 2, and 3 in the E1 mutants were phosphorylated either individually or in the presence of the other sites by the dihydrolipoamide acetyltransferase-protein X-E1 kinase indicating a site-independent mechanism of phosphorylation. Phosphorylation of each site resulted in complete inactivation of the E1. 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-E1-phosphatase resulting in complete reactivation of the E1. The rates of dephosphorylation and reactivation were similar for sites 1, 2, and 3, indicating a random dephosphorylation mechanism.

Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases

“…Figure 3A compares the rates of phosphorylation of the three sites when present individually, demonstrating that the rate of phosphorylation of site 1 was 4.5-fold higher than of site 2 and 16-fold higher than of site 3. The rates of dephosphorylation, on the contrary, appeared to be nearly identical for sites 1, 2 and 3 present either individually or in combinations with the other sites ( Figure 3B), indicating a random mechanism of dephosphorylation of the three sites (Korotchkina and Patel, 1995).…”

“…To distinguish among the three phosphorylation sites, several mutants of recombinant human E1 (replacing serines of the phosphorylation sites with alanine) were generated with a single mutation at site 1 (1A), site 2 (2A) or site 3 (3A); double mutations leaving only one site available for modification, site 1 (2A3A), site 2 (1A3A) or site 3 (1A2A). All mutant E1s were phosphorylated by the E2-E3BP-PDK subcomplex purified from bovine heart (Korotchkina and Patel, 1995). Phosphorylation resulted in concomitant inactivation of E1 as measured by E1 activity in the PDC reaction after reconstitution with E2-E3BP and E3.…”

The relationship between human skeletal muscle pyruvate dehydrogenase phosphatase activity and muscle aerobic capacity

“…Each of these subunits (with the exception of E3BP) is involved in the conversion of pyruvate to acetylCoA in a stepwise manner. The complex is regulated largely via covalent modification by the addition of a phosphate group to at least one of its three serine residues located on the E1␣ subunit of the complex (15,31,33,34,37). Phosphorylation and inactivation are accomplished by a group of specific PDH kinases (PDK1-4), while dephosphorylation and activation are accomplished by a pair of PDH phosphatases (PDP1 and -2; Refs.…”

Nematode pyruvate dehydrogenase kinases: role of the C-terminus in binding to the dihydrolipoyl transacetylase core of the pyruvate dehydrogenase complex

“…Autoradiography of SDS\polyacrylamide gels of rCPDK incubated with [$#P]ATP in the presence and in the absence of native A. suum E1 revealed no evidence for autophosphorylation (Figure 2), in contrast with results reported for recombinant mammalian PDKs [28] but in agreement with results reported recently for rAPDK [20]. However, rCPDK did phosphorylate the native A. suum E1 and generated two phosphorylated E1 bands after autoradiography, as has been observed previously for phosphorylation of the native A. suum E1 [29,30].…”