Isolation of a specific lipoamide dehydrogenase for a branched-chain keto acid dehydrogenase from Pseudomonas putida.
We purified lipoamide dehydrogenase from cells of Pseudomonas putida PpG2 grown on glucose (LPD-glu) and lipoamide dehydrogenase from cells grown on valine (LPD-val), which contained branched-chain keto acid dehydrogenase. LPD-glu had a molecular weight of 56,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and LPD-val had a molecular weight of 49,000. The pH optimum for LPD-glu for reduced nicotinamide adenine dinucleotide oxidation was 7.4, compared with pH 6.5 for LPD-val. When oxidized nicotinamide adenine dinucleotide was included in the assay mixture, the pH optima were 7.1 and 5.7, respectively. There was also a difference in pH optima between the two enzymes for oxidized nicotinamide adenine dinucleotide reduction, but the Michaelis constants and maximum velocities were similar. A purified preparation of branched-chain keto acid dehydrogenase, which was deficient in lipoamide dehydrogenase, was stimulated 10-fold by LPD-val but not by LPD-glu, which suggested that the branched-chain keto acid dehydrogenase of P. putida has a specific requirement for LPD-val. In contrast, a partially purified preparation of 2-ketoglutarate dehydrogenase that was deficient in lipoamide dehydrogenase was stimulated by LPD-glu but not by LPD-val, indicating that this complex has a specific requirement of LPD-glu.
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D008058:Dihydrolipoamide Dehydrogenase / Q000302:isolation & purification*; D005947:Glucose; D006863:Hydrogen-Ion Concentration; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000201:enzymology*; D014633:Valine
D009097:Multienzyme Complexes; D009243:NAD; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D008058:Dihydrolipoamide Dehydrogenase; D014633:Valine; D005947:Glucose