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.

Elusive transition state of alcohol dehydrogenase unveiled

“…The term tunneling and coupled motion has been applied to ADHs and other systems that exhibit 2°KIEs outside the semiclassical range (12,28,37,38), as well as reactions that violate the Rule of the Geometric Mean, as evidenced by inflated mSSEs (1,18,21). The term tunneling and coupled motion appears to be used for both abnormalities in 2°KIEs.…”

“…An elevated mSSE later appeared in other ADHs (19,20) as well as other types of enzymatic hydrogen transfers (21).…”

Optimal alignment for enzymatic proton transfer: Structure of the Michaelis complex of triosephosphate isomerase at 1.2-Å resolution

“…The out-of-plane orientation is assumed to be favorable for extracting the proton and for forming the -orbital of the enediol(ate). At first glance, the structural information suggests a complete proton transfer coordinate from the C1 to the C2 position, with minimal heavy atom motion, and brings to mind the possibility of tunneling of the proton along the complete reaction coordinate (36). Such a streamlined and concerted picture cannot account for a central experimental evidence: any proposed mechanism must also be reconciled with the modest apparent isotope effect for removal of the H1 proton and with the substantial isotope washout when 1-2 H-or 1-3 H-DHAP is used as a substrate.…”

Solvent and Primary Deuterium Isotope Effects Show That Lactate CH and OH Bond Cleavages Are Concerted in Y254F Flavocytochrome b2, Consistent with a Hydride Transfer Mechanism

“…(The solvent isotope effect with deuterated glucose 6-phosphate was not reported in this case.) In general, coupled motion and tunneling have been invoked to explain the decrease in the magnitude of an α-deuterium isotope effect when deuterium replaces the hydrogen being removed in the reaction (35)(36)(37)(38)(39); to our knowledge there have been no other reports of the interplay between solvent and primary isotope effects. The decrease in the primary deuterium isotope effect in D 2 O seen with Y254F flavocytochrome b 2 is much less than that reported for glucose-6-phosphate dehydrogenase.…”