Leucine Dehydrogenase from Corynebacterium pseudodiphtheriticum: Purification and Characterization |
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Authors: | Haruo Misono Kouji Sugihara Yumiko Kuwamoto Shinji Nagata Susumu Nagasaki |
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Institution: | Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Kochi University, Nankoku, Kochi 783 |
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Abstract: | 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. |
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Keywords: | alkyl radicals lipid peroxidation hypoxia cytotoxicity apoptosis |
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