Purification of a branched-chain keto acid dehydrogenase from Pseudomonas putida.
We purified branched-chain keto acid dehydrogenase to a specific activity of 10 mumol/min per mg of protein from Pseudomonas putida grown on valine. The purified enzyme was active with 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate in a ratio of 1.0:0.8:0.7 but showed no activity with either pyruvate or 2-ketoglutarate. There were four polypeptides in the purified enzyme (molecular weights, 49,000, 46,000, 39,000, and 37,000). The purified enzyme was deficient in the specific lipoamide dehydrogenase produced during growth on valine (molecular weight, 49,000). Branched-chain keto acid dehydrogenase required L-valine, oxidized nicotinamide adenine dinucleotide, coenzyme A, thiamine pyrophosphate, and magnesium chloride. A partially purified preparation catalyzed the oxidation of 2-keto-[1-14C]isovalerate to [14C]carbon dioxide, isobutyryl-coenzyme A, and reduced nicotinamide adenine dinucleotide in equimolar amounts. Both the Km and the Vmax for 2-ketoisovalerate were affected by the addition of L-valine to the assay mixture. However, only the Vmax values for oxidized nicotinamide adenine dinucleotide and coenzyme A were affected when L-valine was present. This suggested that valine acted by affecting the binding of branched-chain keto acids to subunit E1 of the complex.
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D002208:Caproates / Q000378:metabolism; D003065:Coenzyme A / Q000378:metabolism; D045782:Hemiterpenes; D007651:Keto Acids / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010421:Pentanoic Acids / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D014633:Valine / Q000494:pharmacology
C021047:3-keto-2-methylvaleric acid; D002208:Caproates; D045782:Hemiterpenes; D007651:Keto Acids; D009097:Multienzyme Complexes; D010421:Pentanoic Acids; D009243:NAD; C001505:alpha-ketoisovalerate; C013082:alpha-ketoisocaproic acid; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D014633:Valine; D003065:Coenzyme A