Alternative pathways and reactions of benzyl alcohol and benzaldehyde with horse liver alcohol dehydrogenase.
Liver alcohol dehydrogenase catalyzes the reaction of NAD+ and benzyl alcohol to form NADH and benzaldehyde by a predominantly ordered reaction. However, enzyme-alcohol binary and abortive ternary complexes form 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 kinetic simulations provide estimates of rate constants for a complete mechanism with the following reactions: (1) E<-->E-NAD+<-->E-NAD(+)-RCH2OH<-->E-NADH-RCHO<-->E-NADH<-->E ; (2) E-NADH<-->E-NADH-RCH2OH<-->E-RCH2OH<-->E; (3) E-NAD+<-->E-NAD(+)-RCHO-->E- NADH-RCOOH<-->E-NADH. The internal equilibrium constant for hydrogen transfer determined at 30 degrees C and pH 7 is about 5:1 in favor of E-NAD(+)-RCH2OH and has a complex pH dependence. Benzyl alcohol binds weakly to free enzyme (Kd = 7 mM) and significantly decreases the rates of binding of NAD+ and NADH. The reaction of NAD+ and benzyl alcohol is therefore kinetically ordered, not random. High concentrations of benzyl alcohol (> 1 mM) inhibit turnover by formation of the abortive E-NADH-RCH2-OH complex, which dissociates at 0.3 s-1 as compared to 6.3 s-1 for E-NADH. The oxidation of benzaldehyde by E-NAD+ (Km = 15 mM, V/E = 0.4 s-1) is inefficient relative to the oxidation of benzyl alcohol (Km = 28 microM, 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 product distributions for the alternative reactions catalyzed by the multifunctional enzyme.
Shearer
G L
GL
Department of Biochemistry, University of Iowa, Iowa City 52242.
Kim
K
K
Lee
K M
KM
Wang
C K
CK
Plapp
B V
BV
D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001547:Benzaldehydes / Q000378:metabolism*; D019905:Benzyl Alcohol; D001592:Benzyl Alcohols / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D009243:NAD / Q000378:metabolism; D013053:Spectrophotometry
D001547:Benzaldehydes; D001592:Benzyl Alcohols; D009243:NAD; D000426:Alcohol Dehydrogenase; D019905:Benzyl Alcohol; C032175:benzaldehyde