KINETICS AND MECHANISM OF ACTIVATED LIVER ALCOHOL DEHYDROGENASE

The mechanism of hydrogen transfer catalyzed by horse liver alcohol dehydrogenase with amidinated amino groups was studied with steady state kinetics. Hydroxybutyrlmidylation of the enzyme increases the maximum velocities of the enzymatic reactions and the rates of dissociation of the enzyme-coenzyme complexes. Primary deuterium isotope effects obtained for oxidation of [1,1-D 2 ]-benzyl alcohol and for reduction of a series of para -substituted benzaldehydes indicate that the turnover numbers reflect the rates of interconversion of central complexes. The magnitudes and signs of the ρ values obtained for oxidation of p -substltuted benzyl alcohols and for reduction of p -substituted benzaldehydes suggest that transfer of hydrogen occurs via concerted hydride and proton transfer in which only a small amount of charge develops in the transition state. The rate of oxidation of benzyl alcohol depends upon a group with a pK of 8.4, which must be unprotonated for maximum activity, but which allows partial activity in its protonated form. The maximum velocity for benzaldehyde reduction catalyzed by native enzyme required protonation of a group with a pK of 8.0. The pH effects for both enzymes can be explained by a coherent model in which a water (or hydroxide) molecule coordinated to the zinc ion acts as a proton donor (or acceptor) and the state of protonation of His-51 modulates the rate of transfer of hydrogen.