Mutations of human class III alcohol dehydrogenase.

Abstract

The residues lining the active site pocket of class III alcohol dehydrogenase differ markedly from those of class I and other classes. Two charged residues in the vicinity of the catalytic site are characteristic of class III. One is Arg115, which is at the outer part of the substrate-binding cleft and is associated with major enzymatic characteristics of class III, i.e. fatty acid activation (Moulis et al., 1991; Holmquist et al., 1993) and glutathione-de-pendent formaldehyde dehydrogenase activity (Engeland et al., 1993). The other charged residue is Asp57, located in the middle part of the substrate pocket. In an attempt to study the relative importance of residues involved in differentiation of classes, we have by site-directed mutagenesis examined not only the function of Asp57, but also the roles of Tyr93 and Thr48 of class III (Estonius et al., 1994). The charge and polarity at positions 57 and 93 were altered by Asp57Leu and Tyr93Phe substitutions, respectively. As opposed to Asp in class III, Leu is found at position 57 in most class I alcohol dehydrogenases, and in contrast to Tyr in class III, Phe or Ala are found at position 93. Therefore, a combination of the Asp57Leu and Tyr93Phe exchanges in one mutant is of interest in order to see if a class I alcohol dehydrogenase substrate pocket, and class I enzymatic properties, can be mimicked by altering the active site structure in a direction toward those of class I enzymes. In addition, to check for differences in the oxidation of short-chain aliphatic alcohols contra S-hy-droxymethylglutathione (HMGSH), we have replaced Thr48 with Ala, a mutation deleterious to class I alcohol dehydrogenase activity (Hoog et al., 1992).