ALTERNATIVE PATHWAYS AND REACTIONS OF BENZYL ALCOHOL AND BENZALDEHYDEWITH HORSE LIVER ALCOHOL-DEHYDROGENASE

Liver alcohol dehydrogenase catalyzes the reaction of NAD+ and benzyl alcohol to form NADH and benzaldehyde by a predominantly ordered react ion. However, enzyme-alcohol binary and abortive ternary complexes for m at high concentrations of benzyl alcohol, and benzaldehyde is slowly oxidized to benzoic acid. Steady-state and transient kinetic studies, equilibrium spectrophotometric measurements, product analysis, and ki netic simulations provide estimates of rate constants for a complete m echanism with the following reactions: (1) E half arrow right over hal f arrow left E-NAD+ half arrow right over half arrow left E-NAD+-RCH2O H half arrow right over half arrow left E-NADH-RCHO half arrow right o ver half arrow left E-NADH half arrow right over half arrow left E; (2 ) E-NADH half arrow right over half arrow left E-NADH-RCH2OH half arro w right over half arrow left E-RCH2OH half arrow right over half arrow left E; (3) E-NAD+ half arrow right over half arrow left E-NAD+-RCHO --> E-NADH-RCOOH half arrow right over half arrow left E-NADH. The int ernal equilibrium constant for hydrogen transfer determined at 30-degr ees-C and pH 7 is about 5:1 in favor of E-NAD+-RCH2OH and has a comple x pH dependence. Benzyl alcohol binds weakly to free enzyme (K(d) = 7 mM) and significantly decreases the rates of binding of NAD+ and NADH. The reaction of NAD+ and benzyl alcohol is therefore kinetically orde red, not random. High concentrations of benzyl alcohol (> 1 mM) inhibi t turnover by formation of the abortive E-NADH-RCH2OH complex, which d issociates at 0.3 s-1 as compared to 6.3 s-1 for E-NADH. The oxidation of benzaldehyde by E-NAD+ (K(m) = 15 mM, V/E = 0.4 s-1) is inefficien t relative to the oxidation of benzyl alcohol (K(m) = 28 muM, V/E = 3. 1 s-1) and leads to a dismutation (2RCHO --> RCH2OH + RCOOH) as E-NADH reduces benzaldehyde. The results provide a description of final prod uct distributions for the alternative reactions catalyzed by the multi functional enzyme.