Isotope effects and structure-reactivity correlations in the yeast alcohol dehydrogenase reaction. A study of the enzyme-catalyzed oxidation of aromatic alcohols

Abstract

Steady-state kinetic parameters for the yeast alcohol dehydrogenase catalyzed oxidation of a series of parasubstituted benzyl alcohols-1, 1-h2 and -1, 1-d2 by NAD+ are reported. Catalytic constants have been found to be characterized by large deuterium isotope effects: kH/kD=4.8, p-Br; 4.2, p-Cl; 3, 4, p-H; 4, 2, p-CH3; 3, 2, p-CH3O. The observed isotope effects on k(cat)/K(A), K(A), and K(B), where K(A) and K(B) are Michaelis constants for NAD+ and alcohol, indicate a borderline rapid equilibrium-steady-state kinetic mechanism involving the random addition of substrate and coenzyme to enzyme. With the exception of p-CH3 and possible p-CH3O substituted benzyl alcohol, k(cat) is concluded to represent a single, rate-limiting hydrogen transfer step. A multiple linear regression analysis of the combined data for benzaldehyde reduction (Klinman, J.P. (1972), J. Biol. Chem. 247, 7977-7987, expanded to include p-CH(CH3) 2-substituted benzaldehyde) and benzyl alcohol oxidation has been carried out to determine the contribution of electronic, hydrophobic, and steric effects to k(cat) and substrate binding. Benzaldehyde binding is concluded to depend on electronic substituent effects as previously reported [log 1/K(ald)=(-0.92 +/- 0.18)sigma+-(0.80 +/- 0.067)], whereas benzyl alcohol binding correlates with substrate hydrophobicity [(log 1/K(alc)=(0.60 +/- 0.14) log P -(1.2 +/- 0.12)]. In the case of benzyl alcohol oxidation, k(cat) is independent of electronic and steric effects; the best of seven equations indicates a small negative dependence of k(cat) on hydrophobicity, which is within experimental error or zero [log k(o)=(-0.075 +/- 0.25) log P -(0.65 +/- 0.19)]. Data for benzaldehyde reduction are correlated at the 99% significance level by a single variable equation [(log k(R)=(2.1 +/- 0.37) sigma+-(0.093 +/- 0.14)] and a two variable equation [(log k(R)=(1.9 +/- 0.33) sigma+ + (0.46 +/- 0.20) log P-(0.46 +/- 0.20)]; these equations indicate (a) a large dependence on electronic substituent as reported previously and (b) a possible role for hydrophobic factors in facilitating catalysis. As the result of the observed hydrophobic substituent effects, different ground-state interactions are suggested for the binding of benzaldehydes and benzyl alcohols. Electronic substituent effects lead to the conclusion that there is little or no change in charge at C-1 of substrate at the transition state, relative to alcohol in the ground state. The significance of these effects to the detailed properties of the hydrogen transfer step is discussed.