Regulation of kinase reactions in pig heart pyruvate dehydrogenase complex.
1. Pig heart pyruvate dehydrogenase complex is inactivated by phosphorylation (MgATP2-) of an alpha-chain of the decarboxylase component. Three serine residues may be phosphorylated, one of which (site 1) is the major inactivating site. 2. The relative rates of phosphorylation are site 1 greater than 2 greater than site 3. 3. The kinetics of the inactivating phosphorylation were investigated by measuring inactivation of the complex with MgATP2-. The apparent Km for the Mg complex of ATP was 25.5 microM; ADP was a competitive inhibitor (Ki 69.8 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 2.8 microM). Inactivation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA. 4. The kinetics of additional phosphorylations (predominantly site 2 under these conditions) were investigated by measurement of 32P incorporation into non-radioactive pyruvate dehydrogenase phosphate containing 3-6% of active complex, and assumed from parrallel experiments with 32P labelling to contain 91% of protein-bound phosphate in site 1 and 9% in site 2. 5. The apparent Km for the Mg complex of ATP was 10.1 microM; ADP was a competitive inhibitor (Ki 31.5 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 1.1 mM). 6. Incorporation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA, although it was less marked at the highest ratios.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D003065:Coenzyme A / Q000494:pharmacology; D004789:Enzyme Activation; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases; D011768:Pyruvate Dehydrogenase Complex / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011773:Pyruvates / Q000494:pharmacology; D013552:Swine
D011768:Pyruvate Dehydrogenase Complex; D011773:Pyruvates; D009243:NAD; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D003065:Coenzyme A