Structural Effects on the Temperature Dependence of Hydride Kinetic Isotope Effects of the NADH/NAD+ Model Reactions in Acetonitrile: Charge-Transfer Complex Tightness Is a Key.

It has recently frequently been found that the kinetic isotope effect (KIE) is independent of temperature (T) in H-tunneling reactions in enzymes but becomes dependent on T in their mutants. Many enzymologists found that the trend is related to different donor-acceptor distances (DADs) at tunneling-ready states (TRSs), which could be sampled by protein dynamics. That is, a more rigid system of densely populated short DADs gives rise to a weaker T dependence of KIEs. Theoreticians have attempted to develop H-tunneling theories to explain the observations, but none have been universally accepted. It is reasonable to assume that the DAD sampling concept, if it exists, applies to the H-transfer reactions in solution, as well. In this work, we designed NADH/NAD+ model reactions to investigate their structural effects on the T dependence of hydride KIEs in acetonitrile. Hammett correlations together with N-CH3/CD3 secondary KIEs were used to provide the electronic structure of the TRSs and thus the rigidity of their charge-transfer complexation vibrations. In all three pairs of reactions, a weaker T dependence of KIEs always corresponds to a steeper Hammett slope on the substituted hydride acceptors. It was found that a tighter/rigid charge-transfer complexation system corresponds with a weaker T dependence of KIEs, consistent with the observations in enzymes.

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

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 ΔEa = EaD - EaH) 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 ΔEa) 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 ΔEa. Overall, the shorter and less broadly distributed DADs resulting from the stronger CT complexation vibrations give rise to a smaller ΔEa. Our results appear to support the explanation that links the T-dependence of KIEs to the donor-acceptor rigidity in enzymes.