Contributions of Valine-292 in the Nicotinamide Binding Site of Liver Alcohol Dehydrogenase and Dynamics to Catalysis,

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Abstract
The participation of Val-292 in catalysis by alcohol dehydrogenase and the involvement of dynamics were investigated. Val-292 interacts with the nicotinamide ring of the bound coenzyme and may facilitate hydride transfer. The substitution of Val-292 with Ser (V292S) increases the dissociation constants for the coenzymes (NAD+ by 50-fold, NADH by 75-fold) and the turnover numbers by 3−7-fold. The V292S enzyme crystallized in the presence of NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol has an open conformation similar to the structure of the wild-type apo-enzyme, rather than the closed conformation observed for ternary complexes with wild-type enzyme. The V292S substitution perturbs the conformational equilibrium of the enzyme and decreases the kinetic complexity, which permits study of the hydride transfer step with steady-state kinetics. Eyring plots show that the ΔH⧧ for the oxidation (V1) of the protio and deuterio benzyl alcohols is 13 kcal/mol and that the kinetic isotope effect of 4.1 is essentially temperature-independent. Eyring plots for the catalytic efficiency for reduction of benzaldehyde (V2/Kp) with NADH or NADD are distinctly convex, being temperature-dependent from 5 to 25 °C and temperature-independent from 25 to 50 °C; the kinetic isotope effect of 3.2 for V2/Kp is essentially independent of the temperature. The temperature dependencies and isotope effects for V1 and V2/Kp are not adequately explained by semiclassical transition state theory and are better explained by hydride transfer occurring through vibrationally assisted tunneling.