Over the past two decades, it has been frequently found that KIEs are temperature(T)-independent with a variety of wild-type enzymes but become T-dependent to different degrees for different variants. Within the contemporary H-tunneling theories, T-independent KIEs have been explained in terms of the narrowly distributed DADs due to a strong enzyme active site compression effect, whereas the strongly T-dependent KIEs in variants correspond to the broadly distributed DADs resulted from the (partial) loss of the dynamical effects from nature. While evidences to support the explanations appear being piled up, use of such KIE tools to evaluate this physical origin for catalysis has, however, been debated. Simulations of the results with other H-transfer/tunneling theories sometimes suggest alternative explanations. We regard that ideas about the correlations of T-dependence of KIEs with DAD sampling in enzymes could be tested by study of the “simpler” reactions in solution, for which DADs could be controlled by structural and solvent effects. Our hypothesis is that a more rigid H-transfer system with less broadly populated DADs gives rise to a weaker T-dependence of KIEs. In this work, we report solvent effects on the T-dependence of KIEs of the NADH/NAD<sup>+</sup> coenzyme analogues to investigate the hypothesis. The effects of solvent polarity and viscosity on the DADs and T-dependence of KIEs were obtained by computation and kinetic experiments. Correlation of DADs with the T-dependence of KIEs was found and our hypothesis is supported. Our results appear to support the proposed role of protein compressive dynamics in short DAD sampling for enzyme catalysis.

Solvent effects on the temperature dependence of kinetic sotope effects in hydride tunneling reactions in solution to test the proposed role of protein dynamics in enzyme catalysis

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