Solvent Effects on the Temperature Dependence of Hydride Kinetic Isotope Effects: Correlation to the Donor–Acceptor Distances

Abstract

Protein structural effects on the temperature (T) dependence of kinetic isotope effects (KIEs) in H-tunneling reactions have recently been used to discuss about the role of enzyme thermal motions in catalysis. Frequently observed nearly T-independent KIEs in the wild-type enzymes and T-dependent KIEs in variants suggest that H-tunneling in the former is assisted by the naturally evolved protein constructive vibrations that help sample short donor–acceptor distances (DADs) needed. This explanation that correlates the T-dependence of KIEs with DAD sampling has been highly debated as simulations following other H-tunneling models sometimes gave alternative explanations. In this paper, solvent effects on the T-dependence of KIEs of two hydride tunneling reactions of NADH/NAD+ analogues (represented by ΔE a = E aD – E aH) were determined in attempts to replicate the observations in enzymes and test the protein vibration-assisted DAD sampling concept. Effects of selected aprotic solvents on the DADPRC’s of the productive reactant complexes (PRCs) and the DADTRS’s of the activated tunneling ready states (TRSs) were obtained through computations and analyses of the kinetic data, including 2° KIEs, respectively. A weaker T-dependence of KIEs (i.e., smaller ΔE a) was found in a more polar aprotic solvent in which the system has a shorter average DADPRC and DADTRS. Further results show that a charge-transfer (CT) complexation made of a stronger donor/acceptor gives rise to a smaller ΔE a. Overall, the shorter and less broadly distributed DADs resulting from the stronger CT complexation vibrations give rise to a smaller ΔE a. Our results appear to support the explanation that links the T-dependence of KIEs to the donor–acceptor rigidity in enzymes.

Correlation of temperature dependence of hydride kinetic isotope effects with donor–acceptor distances in two solvents of different polarities

“…One reason that we observe the same ε-2° KIEs but very different γ-2° KIEs in the two solvents might be that the ε-2° KIE on 3 remote H/D's is less sensitive to the change in DAD than the γ-2° KIE on DMPBIH that involves 6 H/D's being closer to the reaction center. It should be noted that the DAD PRC order in the two solvents that we reported 47 are consistent with the DAD TRS order obtained here. Overall, the results are consistent with our expectations that the positively charged TRS is stabilized and thus more rigid with shorter DAD in the more polar solvent of acetonitrile.…”

“…The observed 2° KIEs that we reported are also listed in the table for comparison. 47 Results show that the signs of the 2° KIEs (normal or inverse) are consistent with the expectations.…”

“…, a more rigid TRS. Since Δ E a of the reaction (1) in acetonitrile is smaller than that in chloroform, 44,47 we expect to see a shorter DAD TRS in acetonitrile than in chloroform. While simulation of a 2° KIE gives rise to an equilibrium (or average) TRS structure from a spectrum of TRS structures sampled, fit of the observed 2° KIEs in the two solutions to the computed TRS structures could give rise to the DAD TRS difference for correlation analysis with the Δ E a 's.…”

“…aUsing the universal solvation model (SMD).bFrom ref. 47 data in parentheses are standard deviations.…”

“…44 We have used the electronic, steric, and solvent effects to control the system rigidity for the study and have found that the hypothesis appears to be supported. 44–47…”