4-Methyl-2-oxopentanoate oxidation by rat skeletal-muscle mitochondria

Abstract

1. Oxidative decarboxylation of 4-methyl-2-oxopentanoate (2-oxoisocaproate) by mitochondria of rat skeletal muscle showed biphasic kinetics. Two apparent Km values of 9.1 micronM and 0.78 mM were established. In broken mitochondria the rate of oxidation was lower and only the higher apparent Km value was found. 2. Isovalerylcarnitine inhibited 4-methyl-2-oxopentanoate oxidation in the presence and absence of carnitine, but isovaleryl-CoA had no inhibitory effect. 3. Addition of ADP enhanced 4-methyl-2-oxopentanolate oxidation. Malate, succinate and 2-oxoglutarate additionally increased the rate of oxidation, but in the absence of ADP succinate and 2-oxoglutarate inhibited. 4. Addition of rotenone and simultaneous addition of carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and valinomycin markedly decreased 4-methyl-2-oxopentanoate oxidation. 5. These observations indicate that the branched-chain 2-oxo acid dehydrogenase complex is situated on the inner side of the mitochondrial inner membrane. 6. In mitochondria and homogenates CO2 was only produced by oxidative decarbosylation of 4-methyl-2-oxopentanoate. In intact muscle oxidation of this oxo acid proceeds more to completeness. 7. The physiological significance of intermediate formation during oxidation of branched-chain amino acids is discussed.