Substituent effects on the ionization step regulating desorption and catalytic oxidation of alcohols bound to liver alcohol dehydrogenase.

1. Transient-state kinetic methods, based on the use of pyrazole as a displacing reagent and reporter ligand, have been applied to examine the pH dependence of rate and equilibrium constants for 2-chloroethanol and 2,2-dichloroethanol binding to the binary complex formed between liver alcohol dehydrogenase and NAD+. 2. The apparent affinity of the enzyme . NAD+ complex for the examined alcohols is dependent on two proton dissociation equilibria. One of these equilibria affects the rate of alcohol association to the binary complex with a ligand-independent pKa value of 7.6, attributable to ionization of zinc-bound water in the enzyme . NAD+ complex. The second proton dissociation equilibrium regulates the rate of alcohol desorption from the enzyme . NAD+ . alcohol complex and exhibits a pKa value of 5.4 and 4.5, respectively, with 2-chloroethanol and 2,2'-dichloroethanol as the ligand. Steady-state kinetic data are reported which indicate that the pKa-5.4-equilibrium controls also the apparent rate of enzymic hydride transfer from 2-chloroethanol to NAD+. 3. These results lend strong support to the mechanism of enzyme action proposed by Kvassman and Pettersson according to which the enzyme . NAD+ . alcohol complex participates in an obligatory proton dissociation step which regulates both desorption and catalytic oxidation of the alcohol ligand. The corresponding pKa value is shown to be lineary dependent on the pKa of the free alcohol ligand with a regression coefficient (Brönstedt alpha value) of about 0.6. The latter observation provides direct evidence that the effect of pH on the reactivity of the enzyme . NAD+ . alcohol complex reflects an alcohol/alcoholate ion equilibration of the enzyme-bound substrate. 4. The particular mode of binding and properties of the active-site zinc ion in liver alcohol dehydrogenase suggest that the catalytic function of the metal ion can be related primarily to facilitation of the process of alcohol oxidation through facilitation of the alcoholate ion formation step now established to precede hydride transfer to NAD+.