Kinetic studies on benzyl alcohol dehydrogenase encoded by TOL plasmid pWWO. A member of the zinc-containing long chain alcohol dehydrogenase family.
The nucleotide sequence of the structural gene for benzyl alcohol dehydrogenase encoded by TOL plasmid pWWO of Pseudomonas putida has been determined. Benzyl alcohol dehydrogenase is a member of the long-chain zinc alcohol dehydrogenase family and, like other alcohol dehydrogenases of this family, contains two zinc atoms per subunit. Benzyl alcohol dehydrogenase, while sharing 31% identical residues with horse liver alcohol dehydrogenase, contains several amino acid substitutions near the active site, some of which may be responsible for the substrate specificity of benzyl alcohol dehydrogenase, which oxidizes exclusively aromatic substrates. Benzyl alcohol dehydrogenase also notably lacks the His51 residue of horse liver alcohol dehydrogenase. Contrary to the results obtained with a mutant human liver alcohol dehydrogenase lacking this residue, the concentration and pKa of solvent proton acceptors had no effect on the catalytic efficiency of benzyl alcohol dehydrogenase. The electronic nature of substituents on the aromatic ring of the substrate influenced the kcat of the enzyme in low concentrations of external proton acceptor, but not in high concentrations. Product inhibition studies demonstrated that benzyl alcohol dehydrogenase followed a general Ordered Bi Bi kinetic mechanism in low proton acceptor conditions, while following a Theorell-Chance kinetic mechanism at high proton acceptor conditions.
Département de Biochimie Médicale, Centre Médicale Universitaire, Geneva, Switzerland.
J P
JP
Shaw
M
M
Rekik
F
F
Schwager
S
S
Harayama
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D007700:Kinetics; D046911:Macromolecular Substances; D008954:Models, Biological; D008969:Molecular Sequence Data; D005810:Multigene Family; D010957:Plasmids; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics*; D015183:Restriction Mapping; D013379:Substrate Specificity; D015032:Zinc / Q000032:analysis / Q000378:metabolism*
D004269:DNA, Bacterial; D046911:Macromolecular Substances; D000429:Alcohol Oxidoreductases; C027016:benzyl alcohol dehydrogenase; D015032:Zinc