Purification and characterization of branched chain alpha-ketoacid dehydrogenase from bovine liver mitochondria.
Branched chain alpha-ketoacid dehydrogenase (EC 1.2.4.3(4)) was solubilized and purified from bovine liver mitochondria for the first time. Decarboxylation of alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and alpha-ketoisocaproate was catalyzed by this multienzyme complex and this activity was co-purified for each substrate. Three enzymatic functions were contained in the complex including decarboxylation of the above ketoacids, transacylation of their simple acid derivatives, and reduction of NAD+ as an overall reaction. Product stoichiometry of these three reactions was 1 CO2:1 acyl-CoA:1 NADH. Activity depended upon the addition of thiamin pyrophosphate, CoASH, and NAD+ which were dissociable cofactors. Physically, two active forms of the enzyme complex were found: a 275,000-dalton unit and a 2 x 10(6)-dalton component. Both showed a characteristic flavin spectra and catalyzed all functions of the complex, implying that 10 small units aggregated into the larger unit. The soluble complex as visualized by electron microscopy had a diameter ranging from 12 to 24 nm corresponding to a molecular weight of 2 x 10(6). The size of the native membrane-bound component remains to be determined.
D000597:Amino Acids, Branched-Chain; D000818:Animals; D002417:Cattle; D007651:Keto Acids; D007658:Ketone Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008854:Microscopy, Electron; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D000597:Amino Acids, Branched-Chain; D007651:Keto Acids; D009097:Multienzyme Complexes; D007658:Ketone Oxidoreductases