Substitution of cysteine-153 ligated to the catalytic zinc in yeast alcohol dehydrogenase with aspartic acid and analysis of mechanisms of related medium chain dehydrogenases

The catalytic zincs in complexes of horse liver and yeast alcohol dehydrogenases (ADH) with NAD(+) and the substrate analogue, 2,2,2-trifluoroethanol, are ligated to two cysteine residues and one histidine residue from the protein and the oxygen from the alcohol. The zinc facilitates deprotonation of the alcohol and is essential for catalysis. In the yeast apoenzyme, the zinc is coordinated to a nearby glutamic acid, which is displaced by the alcohol in the complex with NAD(+). Some homologous medium chain dehydrogenases have a cysteine replaced by aspartic or glutamic acid residues. How an aspartic acid would affect catalysis was studied by replacing Cys-153 in Saccharomyces cerevisiae ADH1 by using site-directed mutagenesis. The C153D enzyme was about as stable as the wild-type enzyme, if EDTA was not included in the buffers. The substitution increased affinity for NAD(+) by 3-fold, but did not affect NADH binding. At pH 7.3, the turnover number for ethanol oxidation (V-1/E-t) decreased by 7-fold and catalytic efficiency decreased 18-fold (V-1/EtKb), but turnover for acetaldehyde reduction (V-2/E-t) was the same as for wild-type enzyme and catalytic efficiency decreased 8-fold (V-2/EtKp). Deuterium isotope effects of 3.0 on V-1/E-t and 3.8 on V-1/EtKb for ethanol oxidation suggest that hydride transfer is more rate-limiting for turnover for the C153D enzyme than by wild-type enzyme. The patterns of pH dependence for V-1/EtKb for ethanol oxidation were similar for both enzymes in the pH range from 7 to 9. The C153D substitution decreased binding of trifluoroethanol by 5-fold and of pyrazole by 65-fold. Substrate specificities for C153D and wild-type ADHs for primary alcohols have similar patterns. Efficiency for secondary alcohols decreased only about 4-fold, and efficiencies for 1,2-propanediol and acetone were about the same as for wild-type enzyme. The C153D substitution modestly affects catalysis by altering ligand exchange on the zinc or local structure. Structures and mechanisms for acid-base catalysis in related medium chain dehydrogenases with substitutions of the homologous cysteine are reviewed and analyzed.