Leucine dehydrogenase from Corynebacterium pseudodiphtheriticum: purification and characterization.
Leucine dehydrogenase [EC 1.4.1.9] was purified to homogeneity from Corynebacterium pseudodiphtheriticum ICR 2210. The enzyme consisted of a single polypeptide with a molecular weight of about 34,000. Stepwise Edman degradation provided the N-terminal sequence of the first 24 amino acids, and carboxypeptidase Y digestion provided the C-terminal sequence of the last 2 amino acids. Although the enzyme catalyzed the reversible deamination of various branched-chain L-amino acids, L-valine was the best substrate for oxidative deamination at pH 10.9 and the saturated concentration. The enzyme, however, had higher reactivity for L-leucine, and the kcat/Km value for L-leucine was higher than that for L-valine. The enzyme required NAD+ as a natural coenzyme. The NAD+ analogs 3-acetylpyridine-NAD+ and deamino-NAD+ were much better coenzymes than NAD+. The enzyme activity was significantly reduced by sulfhydryl reagents and pyridoxal 5'-phosphate. D-Enantiomers of the substrate amino acids competitively inhibited the oxidation of L-valine.
Misono
H
H
Department of Agricultural Chemistry, Kochi University, Nankoku.
Sugihara
K
K
Kuwamoto
Y
Y
Nagata
S
S
Nagasaki
S
S
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D003352:Corynebacterium / Q000201:enzymology*; D004589:Electrophoresis, Disc; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D050803:Leucine Dehydrogenase; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity
D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase