Secondary H/T 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)-2H]-labeled substrate in D2O to slow the chemical steps, relative to product release. After a small correction for the β-equilibrium isotope effect for dehydration of DHAP, the H/T kinetic isotope effect k H/k T = 1.27 ± 0.03 for [1(R)-2H,(S)- 3H]-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 y = 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.