Temperature Dependence of Hydride Kinetic Isotope Effects in Solution to Test the Proposed Role of Protein Dynamics in Enzyme Catalysis - PROJECT SUMMARY Recently proposed protein dynamics coupled to the chemistry of the enzymatic reactions suggests a new possible origin for the enzymatic rate accelerations. Finding such a physical role in catalysis, if any, is of importance to the development of theories for enzyme catalysis that can guide future efforts at design of efficient drugs and biocatalysts. One strategy to study the origin uses enzyme catalyzed H-tunneling reactions that are sensitive to donor-acceptor distances (DADs) and thus to any protein motions that can sample the DADs for H- tunneling to occur. Within the contemporary H-tunneling theories, tunneling of a heavier H isotope requires a shorter DAD, which results in an isotopic rate difference thus a kinetic isotope effect (KIE). As a result, KIE is a function of DAD. Therefore, study of the temperature (T) dependence of KIEs could be used to reflect how enzyme dynamics affect the DAD distributions and thus whether they affect the chemistry of enzymes. Over the past two decades, it has been frequently found that KIEs are T-independent with a variety of wild-type enzymes but become T-dependent to different degrees for different variants. Within those 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 hotly debated. Simulations of the results with other H-transfer/tunneling theories 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 long-term objective is to design H-transfer reactions in solution to replicate the T-dependence of KIEs in enzymes versus variants so as to find whether the KIE observations are caused, or partly caused, by the proposed enzyme’s coupled dynamics. The hypothesis is that a more rigid H-transfer system with less broadly populated DADs gives rise to a weaker T-dependence of KIEs. The specific aims are to use electronic, steric, solvent and remote heavy group vibrational effects to progressively mediate system rigidities to investigate the hypothesis. Hydride transfer reactions of NADH/NAD+ coenzyme analogues will be chosen for the study so that the results can be more directly compared with those from enzymes. Kinetics of the reactions will be determined spectroscopically. Results will provide insight into the argument about whether there is an enzyme active site compression effect. The other significance of the project is that the unprecedented systematic study of the relationship between structure/solvent and T-dependence of KIEs will open a new research direction that could help find appropriate models to describe the hydride tunneling chemistry in both solution and enzymes.
