Effect of pH on coenzyme binding to liver alcohol dehydrogenase.
1. 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. 2. 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. 3. 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.
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D009243:NAD; D010084:Oxidation-Reduction; D011485:Protein Binding
D009243:NAD; D000429:Alcohol Oxidoreductases