Unified mechanism for proton-transfer reactions affecting the catalytic activity of liver alcohol dehydrogenase.

The effect of pH on substrate binding to liver alcohol dehydrogenase has been examined over the pH range 6--10 by transient-state and steady-state kinetic methods. The results provide evidence that there is no significant effect of pH on benzaldehyde binding to the enzyme. Benzyl alcohol association to the binary enzyme . NAD+ complex requires protonation of an ionizing group with a pKa of 7.6 in the binary complex. Substrate dissociation from the enzyme . NAD+ . alcohol complex is regulated by an ionizing group with a pKa of 6.6 (6.4) in the complex formed with naphthyl alcohol (benzyl alcohol). Alcohol desorption from the ternary complex occurs exclusively when the ionizing groups is in the protonated form. A reaction mechanism is proposed which accounts for all major effects of pH on liver alcohol dehydrogenase catalysis over the investigated pH range. The reactivity of the enzyme . NAD+ (enzyme . NAD+ . alcohol) complex is suggested to be regulated by the ionization state of a water (alcohol) molecule bound at the catalytic zinc atom of the enzyme. Zinc-bound water does not function as a binding site for substrates or as a mediator of catalytic proton transfer from substrate to solution at the binary-complex level. Catalytic proton transfer takes place at the ternary-complex level, probably through an alcohol/alcoholate ion interconversion of the enzyme-bound substrate. This proton transfer step can be envisaged to serve the purpose of facilitating hydride transfer during alcohol oxidation and alcohol desorption during aldehyde reduction. The kinetics of proton uptake/release during naphthaldehyde reduction at pH 6 are shown to be consistent with the proposed mechanism of enzyme action.