Clofibric acid and phenylpyruvic acid as biochemical probes for studying soluble bovine liver branched chain ketoacid dehydrogenase.
Purified bovine liver branched chain ketoacid dehydrogenase is inhibited by its reaction products, NADH and the branched chain acyl-CoA thiol esters. This provides an immediate, short term regulation mechanism for this enzyme which commits leucine, isoleucine, and valine to the catabolic pathway. We find that clofibric acid (p-chlorophenoxymethylpropionic acid) and phenylpyruvate also specifically inhibit this multienzyme complex. Fifty percent inhibition occurs at 0.24 mM and 0.40 mM concentrations, respectively. Both compounds bind either free enzyme (E) or the enzyme-substrate (ES) complex to give an abortive enzyme-substrate-inhibitor (ESI) complex. Phenylpyruvate preferentially binds E (Ki = 48 microM) over ES (alpha Ki = 97 microM), while clofibric acid has a reverse preference, binding ES (alpha Ki = 48 microM) with more avidity than E (Ki = 142 microM). Amino acids like L-leucine, beta-leucine, and 2-aminonorbornyl-2-carboxylic acid do not inhibit this enzyme at concentrations as high as 10 mM. These biochemical probes will be useful for studying the catalytic site of this enzyme complex and the consequences of accumulated branched chain ketoacids and their amino acid precursors on muscle and liver metabolism when the dehydrogenase function is impaired.
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000818:Animals; D002417:Cattle; D002994:Clofibrate / Q000494:pharmacology*; D007658:Ketone Oxidoreductases / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D010667:Phenylpyruvic Acids / Q000494:pharmacology*; D013329:Structure-Activity Relationship
D009097:Multienzyme Complexes; D010667:Phenylpyruvic Acids; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D002994:Clofibrate