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 alcoh ol dehydrogenase is biphasic. Solvent isotope effects accompany both phases and are expressed differently at different pressures. These differences al low the extraction of an inverse intrinsic kinetic solvent isotope effect o f 1.1 (i.e., V-D2O/K = 0.9) accompanying hydride transfer and an inverse eq uilibrium 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 cons istent 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 tr ansition-state proton exiting a low-barrier hydrogen bond with a fractionat ion factor between 0.6 and 0.9. The value of the equilibrium effect is cons istent with restrictions of torsional motions of multiple hydrogens of the enzyme protein during the conformational change that accompanies the bindin g of NAD(+). The absence of significant commitments to catalysis accompanyi ng the kinetic solvent isotope effect means that this portion of the proton transfer occurs in the same reactive step as hydride transfer in a concert ed chemical mechanism. The success of this analysis suggests that future me asurements of solvent isotope effects as a function of pressure, in the pre sence of moderate commitments to catalysis, may yield precise estimates of intrinsic solvent isotope effects that are not fully expressed on capture a t atmospheric pressure.