Effect of pH on Coenzyme Binding to Liver Alcohol Dehydrogenase

Abstract

The transient‐state kinetics of ligand‐displacement reactions have been analyzed. Methods based on this analysis have been used to obtain reliable estimates of on‐velocity and off‐velocity constants for coenzyme binding to liver alcohol dehydrogenase at different pH values between 6 and 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 analogues to the enzyme · NAD+ complex. Critical experiments have been 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. Copyright © 1979, Wiley Blackwell. All rights reserved