Jp. Shaw et al., KINETIC-STUDIES ON BENZYL ALCOHOL-DEHYDROGENASE ENCODED BY TOL PLASMID-PWWO - A MEMBER OF THE ZINC-CONTAINING LONG-CHAIN ALCOHOL-DEHYDROGENASE FAMILY, The Journal of biological chemistry, 268(15), 1993, pp. 842-850
The nucleotide sequence of the structural gene for benzyl alcohol dehy
drogenase encoded by TOL plasmid pWWO of Pseudomonas putida has been d
etermined. Benzyl alcohol dehydrogenase is a member of the long-chain
zinc alcohol dehydrogenase family and, like other alcohol dehydrogenas
es 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 sp
ecificity of benzyl alcohol dehydrogenase, which oxidizes exclusively
aromatic substrates. Benzyl alcohol dehydrogenase also notably lacks t
he His51 residue of horse liver alcohol dehydrogenase. Contrary to the
results obtained with a mutant human liver alcohol dehydrogenase lack
ing this residue, the concentration and pK(a) of solvent proton accept
ors had no effect on the catalytic efficiency of benzyl alcohol dehydr
ogenase. The electronic nature of substituents on the aromatic ring of
the substrate influenced the k(cat) of the enzyme in low concentratio
ns of external proton acceptor, but not in high concentrations. Produc
t inhibition studies demonstrated that benzyl alcohol dehydrogenase fo
llowed a general Ordered Bi Bi kinetic mechanism in low proton accepto
r conditions, while following a Theorell-Chance kinetic mechanism at h
igh proton acceptor conditions.