Solvent isotope and mutagenesis studies on the proton relay system in yeast alcohol dehydrogenase 1

Abstract

Alcohol dehydrogenase catalyzes the reversible transfer of a hydride directly from an alcohol to the nicotinamide ring of NAD⁺ to form an aldehyde and NADH, and the proton from the alcohol probably is transferred through a hydrogen-bonded system to the imidazole of His-48. Studies of the pH dependencies, and solvent and substrate isotope effects on the wild-type and the enzyme with His-48 substituted with Gln-48 were used to demonstrate a role for the proton relay system. The H48Q substitution increases affinities for NAD⁺ and NADH by ∼2-fold, suggesting that the overall protein structure is maintained. In contrast, catalytic efficiencies (V/K_m) on ethanol and acetaldehyde and affinity for 2,2,2-trifluoroethanol are decreased by about 10-fold. The pH dependencies for catalytic efficiencies on ethanol and acetaldehyde (log V/K_m versus pH), show pK values of about 7.5 for wild-type enzyme, but ethanol oxidation by H48Q ADH is essentially linear over the pH range from 5.5 to 9.2 with a slope of 0.47. Steady-state kinetics and substrate isotope effects suggest that the kinetic mechanism of H48Q ADH has become partly random for oxidation of ethanol. Both wild-type and H48Q ADHs have pH-independent isotope effects for oxidation (V₁/K_b) of 1-butanol/1-butanol-d₉ of 4, suggesting that hydride transfer is a major rate-limiting step. The pH dependence for butanol oxidation by wild type ADH shows a wavy profile over the pH range from pH 6 to 10, with a ∼2.3-fold larger V₁/K_b in D₂O than in H₂O, an "inverse" isotope effect. The substrate isotope effect of 4 is not altered by the solvent isotope effect, suggesting concerted proton/hydride transfer. The solvent isotope effect can be explained by a ground state with a water bound to the catalytic zinc in the enzyme-NAD⁺ complex, and a transition state that resembles a complex with NADH and aldehyde. In contrast, the H48Q enzyme has a diminished inverse solvent isotope effect of ∼1.3 and an essentially linear pH dependence with a slope of log V₁/K_b against pH of 0.49 for oxidation of 1-butanol, which together are consistent with a transition state where hydroxide ion directly accepts a proton from the 2'-hydroxyl group of the nicotinamide ribose in the proton relay system in the enzyme-NAD⁺-alcohol complex. The results support a catalytic role for His-48 in the proton relay system.

Keywords: Enzyme catalysis; Enzyme kinetics; Mutagenesis; Proton transfer; Transition state.