Partial purification and properties of branched-chain 2-oxo acid dehydrogenase of ox liver.
1. A branched-chain 2-oxo acid dehydrogenase was partially purified from ox liver mitochondria. 2. The preparation oxidized 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutyrate and D- and L-3-methyl-2-oxopentanoate. The apparent Km values for the oxo acids and for thiamin pyrophosphate, CoA, NAD+ and Mg2+ were determined. 3. The oxidation of each oxo acid was inhibited by isovaleryl (3-methylbutyryl)-CoA (competitive with CoA) and by NADH (competitive with NAD+); Ki values were determined. 4. The preparation showed substrate inhibition with each 2-oxo acid. The oxidative decarboxylation of 4-methyl-2-oxo[1-14C]pentanoate was inhibited by 3-methyl-2-oxobutyrate and DL-3-methyl-2-oxopentanoate, but not by pyruvate. The Vmax. with 3-methyl-2-oxobutyrate as variable substrate was not increased by the presence of each of the other 2-oxo acids. 5. Ox heart pyruvate dehydrogenase did not oxidize these branched-chain 2-oxo acids and it was not inhibited by isovaleryl-CoA. The branched-chain 2-oxo acid dehydrogenase activity (unlike that of pyruvate dehydrogenase) was not inhibited by acetyl-CoA. 6. It is concluded that the branched-chain 2-oxo acid dehydrogenase activity is distinct from that of pyruvate dehydrogenase, and that a single complex may oxidize all three branched-chain 2-oxo acids.
D000214:Acyl Coenzyme A / Q000494:pharmacology; D000818:Animals; D002087:Butyrates / Q000378:metabolism; D002417:Cattle; D007651:Keto Acids; D007658:Ketone Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000378:metabolism; D014631:Valerates / Q000378:metabolism
D000214:Acyl Coenzyme A; D002087:Butyrates; D007651:Keto Acids; D014631:Valerates; D009243:NAD; C017447:isovaleryl-coenzyme A; D007658:Ketone Oxidoreductases; D008274:Magnesium; D013835:Thiamine Pyrophosphate