Effect of pressure on deuterium isotope effects of yeast alcohol dehydrogenase: Evidence for mechanical models of catalysis

Moderate pressure accelerates hydride transfer catalyzed by yeast alcohol d ehydrogenase, indicative of a large negative volume of activation [Cho and Northrop (1999) Biochemistry 38,7470-7475]. A comparison of the effects of pressure on the oxidation of normal versus dideuteriobenzyl alcohol generat es a monophasic decrease in the intrinsic isotope effect; therefore, the, v olume of activation for the transition-state of deuteride transfer must be even more negative, by 10.4 mL/mol. This finding appears consistent with hy drogen tunneling previously proposed for this dehydrogenase [Cha, Y., Murra y, C, J., and Klinman, J. P. (1989) Science 243, 1325-1330]. However, a glo bal fit of the primary data shows that the entire isotope effect arises fro m a transition-state phenomenon, unlike normal isotope effects, which arise from different vibrational frequencies in reactant states, and tunneling i sotope effects, which arise from a mixture of both states. Assuming the phe nomenon is tunneling, the isotopic data are consistent with a Bell tunnelin g correction factor of Q(H) = 12 and an imaginary frequency of nu(H)(double dagger) = 1220 cm(-1), the first so calculated from experimental enzymatic data. This excessively large correction factor and the large difference in the isotopic activation volumes, plus the low isotope effects at extrapola ted pressures, challenge traditional applications of physical organic chemi stry and transition-state theory to enzymatic catalysis. They suggest inste ad that something other than transition-state stabilization or tunneling is responsible for the rate acceleration, something unique to the enzymatic t ransition state that does not occur in nonenzymatic reactions. Arguments fo r the vibrational model of coupled atomic motions and the fluctuating enzym e model of protein domain motion are put forward as possible interpretation s.