Effect of pH on the Binding of Decanoate and Trifluoroethanol to Liver Alcohol Dehydrogenase

1 Rate constants for dissociation of the substrate analogues decanoate and trifluoroethanol from the respective ternary complex formed with liver alcohol dehydrogenase and NAD+ have been determined at different pH. The rate of decanoate dissociation exhibits no pronounced dependence on pH. The trifluoroethanol dissociation rate is regulated by a proton equilibrium with a pKa of 4.3, alcohol desorption occurring exclusively from the protonated form of the ternary complex. 2 In contrast to what has been previously suggested from equilibrium binding studies, these observations provide evidence that the two substrate analogues combine to the same, protonated, form of the binary enzyme · NAD+ complex. This means that catalytic proton release during the enzymic oxidation of alcohol substrates is likely to take place at the ternary-complex level. It is shown that proton release coupled to the catalytic hydride transfer step can be readily detected at the appropriate pH during the enzymic oxidation of benzyl alcohol. 3 The pKa-4.3 dependence of trifluoroethanol dissociation is suggested to reflect an alcohol/alcoholate ion equilibration of the enzyme-bound substrate analogue. Substitution of trifluoroethanol by benzyl alcohol in the enzyme · NAD+· alcohol complex results in an uptake of close to one proton per active site of the enzyme at pH 5.7. This observation is consistent with our previous proposal that the enzyme · NAD+· benzyl alcohol complex participates in a similar alcohol/alcoholate ion equilibrium with a pKa of 6.4.