RESIDUES SPECIFIC FOR CLASS-III ALCOHOL-DEHYDROGENASE - SITE-DIRECTEDMUTAGENESIS OF THE HUMAN ENZYME

Human class III alcohol dehydrogenase (with both glutathione-dependent formaldehyde dehydrogenase and alcohol dehydrogenase activities) was expressed, and studied by site-directed mutagenesis corresponding to t hree amino acid residues that are affecting the substrate-binding pock et of class I(with alcohol dehydrogenase activity only). A Thr48Ala ex change results in an enzyme essentially without alcohol dehydrogenase activity but with some glutathione-dependent formaldehyde dehydrogenas e activity retained. This indicates that coordination to the enzyme of S-hydroxymethylglutathione is mediated by interactions additional to, or different from, those utilized for primary and secondary alcohols. An Asp57Leu mutation causes considerable loss of the formaldehyde deh ydrogenase activity, showing that a negative charge at position 57 is a prerequisite for this class III-type of activity, in the same manner as a positive charge at position 115 has been previously demonstrated to be crucial. Therefore, Asp57 and Arg115 appear to contribute equal ly to the interactions with S-hydroxymethylglutathione, compatible wit h defining the class III-type of specificity and possibly explaining t he dependence on glutathione. A Tyr93Phe mutant exhibits decreased k(c at) values for substrates in general and correlates with inhibition of alcohol dehydrogenase activity by 4-methylpyrazole, a potent inhibito r of the class I enzymes. In a double mutant, Asp57Leu/Tyr93Phe, the e ffects of the two mutations are potentiating one another, yielding a f all in k(cat)/K-m for hydroxymethylglutathione by a factor of 1250, i. e., a still further loss of class III-type activity. At the same time, the alcohol dehydrogenase activity of Asp57Leu/Tyr93Phe has gained a characteristic class I property, complete inhibition by 4-methylpyrazo le at concentrations only partially reducing the activity of the wild- type class III enzyme.