title: Secondary H/T and D/T isotope effects in enzymatic enolization reactions. Coupled motion and tunneling in the triosephosphate isomerase reaction.
abstract: Secondary kH/kT kinetic isotope effects in H2O and kH/kT or kD/kT 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 beta-equilibrium isotope effect for dehydration of DHAP, the H/T kinetic isotope effect kH/kT = 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, KH/KT = 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701, USA.
authors.forename: W C
authors.initials: WC
authors.lastname: Alston
authors.forename: M
authors.initials: M
authors.lastname: Kanska
authors.forename: C J
authors.initials: CJ
authors.lastname: Murray
mesh_terms: D000818:Animals; D001665:Binding Sites; D003903:Deuterium; D004099:Dihydroxyacetone Phosphate / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D006859:Hydrogen; D007700:Kinetics; D008956:Models, Chemical; D008968:Molecular Conformation; D015394:Molecular Structure; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D013816:Thermodynamics; D014305:Triose-Phosphate Isomerase / Q000378:metabolism*; D014316:Tritium
chemical_list: D005656:Fungal Proteins; D014316:Tritium; D005986:Glyceraldehyde 3-Phosphate; D004099:Dihydroxyacetone Phosphate; D006859:Hydrogen; D003903:Deuterium; D014305:Triose-Phosphate Isomerase