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 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.