Abstract

Homogeneous class II alcohol dehydrogenase (pi-ADH) has been isolated from human liver homogenates by chromatography on DE-52 cellulose, 4-[3-[N-(6-amino-caproyl)amino]propyl]pyrazole-Sepharose, SP-Sephadex C-50, and agarose-hexane-AMP, yielding an enzyme that has a significantly higher specific activity and is markedly more stable than that isolated by an earlier procedure. pi-ADH is composed of two identical 40 000-dalton subunits, contains 4 mol of zinc/dimer, and is readily inhibited by metal-chelating agents. The purified enzyme binds two molecules of coenzyme per dimer, exhibits an absorption maximum at 280 nm, epsilon 280 = 57 000, and exhibits an isoelectric point of 8.6. The class II isozyme catalyzes the oxidation of a variety of alcohols with Km values ranging from 7 microM to 560 mM and with kcat values from 32 min-1 to 600 min-1 and demonstrates a preference for hydrophobic substrates. The kcat/Km ratio for ethanol oxidation exhibits a pH maximum at 10.4.