Secondary WT and D/T isotope effects in enzymatic enolization reactions. Coupled motion and tunneling in the triosephosphate isomerase reaction

Secondary k(H)/k(T) kinetic isotope effects in H2O and k(H)/k(T) or k(D)/k(T) isotope effects in D2O have been measured for the triosephosphate isomerase-catalyzed conversion of dihydroxyacetone 3-phosphate (DHAP) to D-glyceraldehyde 3-phosphate. The proton transfer steps are made rate-limiting using [1(R)-H-2]-labeled substrate in D2O to slow the chemical steps, relative to product release. After a small correction for the beta-equilibrium isotope effect for dehydration of DHAP, the WT kinetic isotope effect k(H)/k(T) + 1.27 +/- 0.03 for [1(R)-H-2,(S)-H-3]-labeled substrate in D2O is subtantially larger than the equilibrium isotope effect for enolization of DHAP, K-H/K-T = 1.12 The H/T isotope effect is related to the D/T isotope effect with a Swain-Schaad exponent gamma = 4.4 +/- 1.3. These results are consistent with coupled motion of the C-1 primary and secondary hydrogens of DHAP and tunneling. Large secondary kinetic isotope effects are a general feature of enzymatic enolization reactions while nonenzymatic enolization reactions show secondary kinetic isotope effects that are substantially smaller than equilibrium effects [Alston, W. A., II, Haley, K., Kanski, R., Murray, C. J., & Pranata, J. (1996) J. Am. Chem Soc., 118, 6562-6569]. Possible origins for these differences in transition state structure are discussed.