Studies of NADP(+)-preferred secondary alcohol dehydrogenase from Thermoanaerobium brockii.
Alcohol dehydrogenase has been purified from the cell-free preparation of Thermoanaerobium brockii to homogeneity, employing combined DEAE, Sephadex, and affinity chromatographic procedures. The enzyme is tetrameric having subunit molecular weight of 40.4 x 10(3). The purified alcohol dehydrogenase is capable of utilizing either NAD+ or NADP+ to oxidize primary and secondary alcohols, although it prefers NADP+ as the coenzyme and secondary alcohols as substrates. Inactivation of the enzymic activity by sensitized photooxidation and carboxymethylation implicates the presence of catalytically important histidine and cysteine residues. Kinetic studies indicate that Thermoanaerobium alcohol dehydrogenase catalyzes NADP(+)-linked oxidations of secondary alcohols by an ordered bi-bi mechanism with NADP+ as the leading reactant. The preference of the Thermoanaerobium enzyme for NADP+ is correlated with its low dissociation constants (KA and KiA) and high turnover rate (V/Et). The corresponding kinetic parameters also contribute to the preference of this enzyme for secondary alcohols.
Ottawa-Carleton Chemistry Institute, Carleton University, Ont., Canada.
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al-Kassim
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Tsai
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009243:NAD / Q000502:physiology; D009249:NADP / Q000502:physiology*; D013379:Substrate Specificity
D046911:Macromolecular Substances; D009243:NAD; D009249:NADP; D000429:Alcohol Oxidoreductases; C025192:isopropanol dehydrogenase (NADP)