Substitution of both histidines in the active site of yeast alcohol dehydrogenase 1 exposes underlying pH dependencies.

Histidine residues 44 and 48 in yeast alcohol dehydrogenase (ADH) bind to the coenzymes NAD(H) and contribute to catalysis. The individual H44R and H48Q substitutions alter the kinetics and pH dependencies, and now the roles of other ionizable groups in the enzyme were studied in the doubly substituted H44R/H48Q ADH. The substitutions make the enzyme more resistant to inactivation by diethyl pyrocarbonate, modestly improve affinity for coenzymes, and substantially decrease catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The pH dependencies for several kinetic parameters are shifted from pK values for wild-type ADH of 7.3-8.1 to values for H44R/H48Q ADH of 8.0-9.6, and are assigned to the water or alcohol bound to the catalytic zinc. It appears that the rate of binding of NAD+ is electrostatically favored with zinc-hydroxide whereas binding of NADH is faster with neutral zinc-water. The pH dependencies of catalytic efficiencies (V/EtKm) for ethanol oxidation and acetaldehyde reduction are similarly controlled by deprotonation and protonation, respectively. The substitutions make an enzyme that resembles the homologous horse liver H51Q ADH, which has Arg-47 and Gln-51 and exhibits similar pK values. In the wild-type ADHs, it appears that His-48 (or His-51) in the proton relay systems linked to the catalytic zinc ligands modulate catalytic efficiencies.

Specific base catalysis by yeast alcohol dehydrogenase I with substitutions of histidine-48 by glutamate or serine residues in the proton relay system.

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.