초록
His-48 in yeast alcohol dehydrogenase I (His 51 in horse liver alcohol dehydrogenase) is a highly conserved residue in the active sites of many alcohol dehydrogenases. The imidazole group of His-48 may participate in base catalysis of proton transfer as it is linked by hydrogen bonds through the 2'-hydroxyl group of the nicotinamide ribose and the hydroxyl group of Thr-45 to the hydroxyl group of the alcohol bound to the catalytic zinc. In this study, His-48 was substituted with a glutamic acid residue to determine if a carboxylate could replace imidazole or to a serine residue to determine if the exposure of the 2'-hydroxyl group of the ribose to solvent would allow proton transfer to water without base catalysis. At pH 7.3, the H48E substitution increases affinity for NAD+ and NADH 17- or 2.6-fold, but decreases catalytic efficiency (V/Km) on ethanol by 70-fold and on acetaldehyde by 6-fold relative to wild-type enzyme. The H48S substitution increases affinity for coenzymes by 2-fold and decreases (V/Km) on ethanol and acetaldehyde only by ∼3-fold. The substituted enzymes show substrate deuterium isotope (H/D) effects of 3-4 for turnover number (V1) and catalytic efficiency (V1/Kb) for ethanol oxidation, indicating that hydrogen transfer is partially rate-limiting and suggesting a somewhat more random mechanism for binding of ethanol and NAD+. For reduction of acetaldehyde, the deuterium isotope effects are small, and the kinetic mechanism appears to be ordered for binding of NADH first and acetaldehyde next. The pH dependencies for H48E and H48S ADHs can be described by a mechanism with pK values of about 6-7 and 9. However, the pH dependencies for oxidation of ethanol and butanol by the H48S enzyme are also simply described by a straight line, with slopes of log V1/Kb against pH of 0.37 or 0.43, respectively. The linear dependence apparently represents catalysis by hydroxide that has a low activity coefficient due to the protein environment, or to a kinetically complex proton transfer. The effects of the substitutions of His-48 show that this residue contributes to catalysis, although many dehydrogenases also have other residues.