Effect of pH on Coenzyme Binding to Liver Alcohol Dehydrogenase

Abstract

The transient-state kinetics of ligand-displacement reactions were analyzed. Methods based on this analysis were used to obtain reliable estimates of on- and off-velocity constants for coenzyme binding to [horse] liver alcohol dehydrogenase at different pH values from 6-10. The rate of NADH dissociation from the enzyme shows no pronounced dependence on pH. The rate of NAD+ dissociation is controlled by a group with a pKa of 7.6, agreeing with the pKa reported to regulate the binding of certain inhibitory substrate analogs to the enzyme.cntdot.NAD+ complex. Critical experiments were performed to test a recent proposal that on-velocity constants for the binding of NADH and NAD+ are controlled by proton equilibria exhibiting different pKa values. The results show that association rates for NADH and NAD+ exhibit the same pH dependence corresponding to a pKa of 9.2. Titrimetric evidence is presented indicating that the latter effect of pH derives from ionization of a group which affects the anion-binding capacity of the coenzyme-binding site.