title: Rat liver alcohol dehydrogenase of class III. Primary structure, functional consequences and relationships to other alcohol dehydrogenases.
abstract: The amino acid sequence of alcohol dehydrogenase of class III from rat liver (the enzyme ADH-2) has been determined. This type of structure is quite different from those of both the class I and the class II alcohol dehydrogenases. The rat class III structure differs from the rat and human class I structures by 133-138 residues (exact value depending on species and isozyme type); and from that of human class II by 132 residues. In contrast, the rat/human species difference within the class III enzymes is only 21 residues. The protein was carboxymethylated with iodo[2(14)C]acetate, and cleaved with CNBr and proteolytic enzymes. Peptides purified by exclusion chromatography and reverse-phase high-performance liquid chromatography were analyzed by degradation with a gas-phase sequencer and with the manual 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method. The protein chain has 373 residues with a blocked N terminus. No evidence was obtained for heterogeneity. The rat ADH-2 enzyme of class III contains an insertion of Cys at position 60 in relation to the class I enzymes, while the latter alcohol dehydrogenase in rat (ADH-3) has another Cys insertion (at position 111) relative to ADH-2. The structure deduced explains the characteristic differences of the class III alcohol dehydrogenase in relation to the other classes of alcohol dehydrogenase, including a high absorbance, an anodic electrophoretic mobility and special kinetic properties. The main amino acid substitutions are found in the catalytic domain and in the subunit interacting segments of the coenzyme-binding domain, the latter explaining the lack of hybrid dimers between subunits of different classes. Several substitutions provide an enlarged and more hydrophilic substrate-binding pocket, which appears compatible with a higher water content in the pocket and hence could possibly explain the higher Km for all substrates as compared with the corresponding values for the class I enzymes. Finally the class III structure supports evolutionary relationships suggesting that the three classes constitute clearly separate enzymes within the group of mammalian zinc-containing alcohol dehydrogenases.
authors.affiliation: Department of Chemistry I, Karolinska Institutet, Stockholm, Sweden.
authors.forename: P
authors.initials: P
authors.lastname: Julià
authors.forename: X
authors.initials: X
authors.lastname: Pareś
authors.forename: H
authors.initials: H
authors.lastname: Jörnvall
mesh_terms: D000429:Alcohol Oxidoreductases / Q000032:analysis* / Q000145:classification; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D003067:Coenzymes / Q000032:analysis; D006736:Horses; D006801:Humans; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D051381:Rats; D013045:Species Specificity; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D003067:Coenzymes; D010455:Peptides; D000429:Alcohol Oxidoreductases