TI - Site specificity of four pyruvate dehydrogenase kinase isoenzymes toward the three phosphorylation sites of human pyruvate dehydrogenase . AB - Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation-dephosphorylation of three specific serine residues ( site 1 , Ser-264 ; site 2 , Ser-271 ; site 3 , Ser-203 ) of the alpha subunit of the pyruvate dehydrogenase (E1) component . Phosphorylation is carried out by four pyruvate dehydrogenase kinase ( PDK ) isoenzymes . Specificity of the four mammalian PDKs toward the three phosphorylation sites of E1 was investigated using the recombinant E1 mutant proteins with only one functional phosphorylation site present . All four PDKs phosphorylated site 1 and site 2 , however , with different rates in phosphate buffer ( for site 1 , PDK2 &gt ; PDK4 approximately PDK1 &gt ; PDK3 ; for site 2 , PDK3 &gt ; PDK4 &gt ; PDK2 &gt ; PDK1 ) . Site 3 was phosphorylated by PDK1 only . The maximum activation by dihydrolipoamide acetyltransferase was demonstrated by PDK3 . In the free form , all PDKs phosphorylated site 1 , and PDK4 had the highest activity toward site 2 . The activity of the four PDKs was stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2 . Substitution of the site 1 serine with glutamate , which mimics phosphorylation -dependent inactivation of E1 , 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 distribution could contribute to the tissue -specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states .