Effects of high pressure on solvent isotope effects of yeast alcohol dehydrogenase

The effect of pressure on the capture of a substrate alcohol by yeast alcohol dehydrogenase is biphasic. Solvent isotope effects accompany both phases and are expressed differently at different pressures. These differences allow the extraction of an inverse intrinsic kinetic solvent isotope effect of 1.1 (i.e., V-D2O/K = 0.9) accompanying hydride transfer and an inverse equilibrium solvent isotope effect of 2.6 (i.e., K-D2O(S) = 0.4) accompanying the binding of nucleotide, NAD(+). The Value of the kinetic effect is consistent with a reactant-state E-NAD(+)-Zn-OH2 having a fractionation factor of phi approximate to 0.5 for the zinc-bound water in conjunction with a transition-state proton exiting a low-barrier hydrogen bond with a fractionation factor between 0.6 and 0.9. The value of the equilibrium effect is consistent with restrictions of torsional motions of multiple hydrogens of the enzyme protein during the conformational change that accompanies the binding of NAD(+). The absence of significant commitments to catalysis accompanying the kinetic solvent isotope effect means that this portion of the proton transfer occurs in the same reactive step as hydride transfer in a concerted chemical mechanism. The success of this analysis suggests that future measurements of solvent isotope effects as a function of pressure, in the presence of moderate commitments to catalysis, may yield precise estimates of intrinsic solvent isotope effects that are not fully expressed on capture at atmospheric pressure.