Abstract
A significant contemporary question in enzymology involves the role of protein dynamics and hydrogen tunneling in enhancing enzyme catalyzed reactions. Here, we report a correlation between the donor-acceptor distance (DAD) distribution and intrinsic kinetic isotope effects (KIEs) for the dihydrofolate reductase (DHFR) catalyzed reaction. This study compares the nature of the hydride-transfer step for a series of active-site mutants, where the size of a side chain that modulates the DAD (I14 in E. coli DHFR) is systematically reduced (I14V, I14A, and I14G). The contributions of the DAD and its dynamics to the hydride-transfer step were examined by the temperature dependence of intrinsic KIEs, hydride-transfer rates, activation parameters, and classical molecular dynamics (MD) simulations. Results are interpreted within the framework of the Marcus-like model where the increase in the temperature dependence of KIEs arises as a direct consequence of the deviation of the DAD from its distribution in the wild type enzyme. Classical MD simulations suggest new populations with larger average DADs, as well as broader distributions, and a reduction in the population of the reactive conformers correlated with the decrease in the size of the hydrophobic residue. The more flexible active site in the mutants required more substantial thermally activated motions for effective H-tunneling, consistent with the hypothesis that the role of the hydrophobic side chain of I14 is to restrict the distribution and dynamics of the DAD and thus assist the hydride-transfer. These studies establish relationships between the distribution of DADs, the hydride-transfer rates, and the DAD's rearrangement toward tunneling-ready states. This structure-function correlation shall assist in the interpretation of the temperature dependence of KIEs caused by mutants far from the active site in this and other enzymes, and may apply generally to C-H→C transfer reactions.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.