Solvent deuterium isotope effect on the liver alcohol dehydrogenase reaction

Previous work on the kinetic mechanism of the liver alcohol dehydrogenase (LADH) catalyzed reduction of aromatic aldehydes by NADH has established that the transient kinetic reaction under conditions where enzyme is limited to a single reaction is biphasic and that the rapid transient shows a primary isotope effect when NADD is used. Furthermore, solution of the rate equations for reduction of aromatic aldehydes has established that this rapid transient is determined by a single rate constant for ternary complex interconversion (i.e., the chemical step). We have determined the deuterium solvent isotope effect on the rapid transient observed during aromatic aldehyde reduction. The isotope effect is 1.0 +- 0.1, indicating that the movement of proton is uncoupled from the addition of hydride to aldehyde carbonyl. Furthermore, we observe inverse isotope effects (k/sub D2O//k/sub H2O/ = 2-3) on the slow transient observed during aldehyde reduction; the rate-limiting step for this reaction has been established to be alcohol dissociation from the ternary complex. In addition, the solvent isotope effect on the transient kinetic rate of alcohol oxidation is also inverse as is the isotope effect on the rate of phenanthroline binding to active-site zinc. The pH dependence of these solvent isotope effects is in accord with proton transfer uncoupled from the rate-limiting process. The pK/sub a/ of the enzyme base catalytic group is perturbed by approx. 0.3 to 0.5 pK/sub a/ unit in D2O and there is no solvent isotope effect at equivalent pH(D) values on the pH(D) dependence curves. These data and most of the other data collected on the chemical steps for LADH reaction are consistent with a modification of a recent proposal for LADH catalysis in which aldehyde binds to the active-site zinc and the zinc-bound water molecule serves as an acid-base catalyst