Effect of pressure on a heavy-atom isotope effect of yeast alcohol dehydrogenase.

Hydrostatic pressure causes a monophasic decrease in the (13)C primary isotope effect expressed on the oxidation of benzyl alcohol by yeast alcohol dehydrogenase. The primary isotope effect was measured by the competitive method, using whole-molecule mass spectrometry. The effect is, therefore, an expression of isotopic discrimination on the kinetic parameter V/K, which measures substrate capture. Moderate pressure increases capture by activating hydride transfer, the transition state of which must therefore have a smaller volume than the free alcohol plus the capturing form of enzyme [Cho, Y.-K.; Northrop, D. B. Biochemistry 1999, 38, 7470-7475]. The decrease in the (13)C isotope effect with increasing pressure means that the transition state for hydride transfer from the heavy atom must have an even smaller volume, measured here to be 13 mL.mol(-1). The pressure data factor the kinetic isotope effect into a semiclassical reactant-state component, with a null value of k(12)/k(13) = 1, and a transition-state component of Q(12)/Q(13) = 1.028 (borrowing Bell's nomenclature for hydrogen tunneling corrections). A similar experiment involving a deuterium isotope effect previously returned the same volume and null value, plus a pressure-sensitive isotope effect [Northrop, D. B.; Cho, Y.-K. Biochemistry 2000, 39, 2406-2412]. Consistent with precedence in the chemical literature, the latter suggested a possibility of hydrogen tunneling; however, it is unlikely that carbon can engage in significant tunneling at ambient temperature. The fact that the decrease in activation volumes for hydride transfer is equivalent when one mass unit is added to the carbon end of a scissile C-H bond and when one mass unit is added to the hydrogen end is significant and suggests a common origin.