Site specificity of four pyruvate dehydrogenase kinase isoenzymes toward the three phosphorylation sites of human pyruvate dehydrogenase

Activity of the mammalian pyruvate dehydrogenase complex is regulated by ph osphorylation-dephosphorylation of three specific serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the alpha subunit of the pyru vate dehydrogenase (EI) component. Phosphorylation is carried out by four p yruvate dehydrogenase kinase (PDK) isoenzymes. Specificity of the four mamm alian PDKs toward the three phosphorylation sites of E1 was investigated us ing the recombinant E1 mutant proteins with only one functional phosphoryla tion site present. All four PDKs phosphorylated site 1 and site 2, however, with different rates in phosphate buffer (for site 1, PDK2 > PDK4 approxim ate to PDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1). Site 3 was phos phorylated by PDK1 only. The maximum activation by dihydrolipoamide acetylt ransferase was demonstrated by PDK3. In the free form, all PDKs phosphoryla ted site 1, and PDK4 had the highest activity toward site 2. The activity o f the four PDKs was stimulated to a different extent by the reduction and a cetylation state of the lipoyl moieties of dihydrolipoamide acetyltransfera se with the maximum stimulation of PDK2. Substitution of the site I serine with glutamate, which mimics phosphorylation-dependent inactivation of El, did not affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1 . Site specificity for phosphorylation of four PDKs with unique tissue dist ribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states.