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.
