Purification and characterization of human liver branched-chain alpha-keto acid dehydrogenase complex.
Human liver BCKADH complex was purified. On SDS-polyacrylamide gel electrophoresis, the purified enzyme complex gave three major bands having molecular weights of 51,000, 46,000, and 36,000, and one minor band with a molecular weight of 55,000. The minor band corresponded in molecular weight to lipoamide oxidoreductase which was purified separately. The purified BCKADH represented only approximately 20% of the maximum activity when assayed without addition of exogenous lipoamide oxidoreductase, indicating that lipoamide oxidoreductase component was readily dissociable from the complex. The BCKADH effectively oxidized all of KIV, KIC, and KMV, yielding apparent Km values in the range of 14-17 microM for those alpha-keto acids. Vmax values obtained were 0.86, 0.61, and 0.51 mumole NADH produced/min/mg of protein for KIV, KIC, and KMV, respectively, in the presence of excess amount of lipoamide oxidoreductase. This ratio of Vmax values was practically identical to those of specific activities obtained with respective branched-chain alpha-keto acids at each purification step. The enzyme complex also oxidized pyruvate and alpha-ketoglutarate to a lesser extent. Kinetic experiments gave Km values of 0.98 and 2.9 mM for pyruvate and alpha-ketoglutarate, respectively, with Vmax of 0.43 and 0.08 mumole NADH produced/min/mg of protein. NAD and CoASH were absolutely required for the reaction. Km values for NAD and CoASH were estimated to be 47 and 25 microM, respectively.
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007658:Ketone Oxidoreductases / Q000302:isolation & purification* / Q000502:physiology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000502:physiology; D011487:Protein Conformation; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)