Mechanism of activation of bovine kidney pyruvate dehydrogenase a kinase by malonyl-CoA and enzyme-catalyzed decarboxylation of malonyl-CoA.
The activity of the pyruvate dehydrogenase kinase, which phosphorylates and thereby inactivates the pyruvate dehydrogenase complex, was stimulated by malonyl-CoA. Treatment with [2-14C]malonyl-CoA resulted in acylation of sites in the complex. Both acylation and activation of kinase activity increased in a time-dependent manner with a parallel increase in those activities when the malonyl-CoA:CoA ratio was varied. Protein-bound acyl groups were labilized by performic acid treatment indicating their attachment to protein at thiol residues; however, the product released was volatile, which is not characteristic of malonic acid. While malonyl-CoA was initially free of acetyl-CoA, stimulation of kinase activity and acylation of sites in the complex by malonyl-CoA were shown to be contingent upon enzyme-catalyzed decarboxylation. Decarboxylation appeared to be catalyzed by a trace contaminant present in highly purified preparations of both the pyruvate and 2-oxoglutarate dehydrogenase complexes. Under conditions in which both free CoA was removed (by conversion to succinyl-CoA) and then, after various periods, free acetyl-CoA was removed (by enzymic conversion to acetyl phosphate), both acetylation of sites in the complex and activation of kinase activity increased in a time-dependent manner. Concomitantly there was a decrease in the concentration dependence for activation of the kinase by malonyl-CoA. Our results strongly support the conclusion that activation of kinase activity is associated with acylation of sites in the complex, and that, in the case of malonyl-CoA, those processes depend on enzyme-catalyzed decarboxylation.
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism*; D000215:Acylation; D000818:Animals; D002384:Catalysis; D002417:Cattle; D003653:Decarboxylation; D004789:Enzyme Activation; D005561:Formates; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000378:metabolism* / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism
D000214:Acyl Coenzyme A; D005561:Formates; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex; C008817:peroxyformic acid; D008316:Malonyl Coenzyme A; D000105:Acetyl Coenzyme A; D007655:Ketoglutarate Dehydrogenase Complex; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases