The overall objective of these projects is to illuminate the role of hydrogen bonding, hydrogen tunneling, and protein motion, as well as the impact of distal mutations, in enzyme reactions. Movies of hydrogen tunneling in LADH and DHFR are available below.
These simulations suggested the concepts of a free energy landscape for enzyme catalysis and a network of coupled equilibrium motions that facilitates enzyme catalysis.
We have developed a hybrid quantum/classical molecular dynamics approach for simulating proton and hydride transfer reactions in enzymes.41, 46, 48 This hybrid approach includes electronic and nuclear quantum effects, as well as the motion of the entire solvated enzyme. The methodology provides detailed mechanistic information at the molecular level and allows the calculation of rate constants and kinetic isotope effects. It also enables us to investigate the relation between enzyme motion and activity.
We applied this hybrid approach to hydride transfer in LADH.
LADH Movies of H Tunneling
We applied this hybrid quantum/classical molecular dynamics approach to hydride transfer in DHFR. Some of this work was done in collaboration with the Benkovic group at Penn State. An analysis of the simulations led to the identification and characterization of a network of coupled equilibrium motions that extends throughout the enzyme and represents conformational changes that facilitate the charge transfer process. Mutations distal to the active site were shown to significantly impact the catalytic rate constant by altering the conformational motions of the entire enzyme and thereby changing the probability of sampling conformations conducive to the catalyzed reaction. In addition, we developed a computational approach for ranking mutant enzymes according to the catalytic reaction rates and applied this approach to a series of 15 DHFR mutants.
DHFR Movies of H Tunneling
We applied this hybrid quantum/classical molecular dynamics approach to the two proton transfer reactions catalyzed by KSI.121 The results suggest that relatively small conformational changes of the enzyme active site and substrate strengthen the hydrogen bonds that stabilize the intermediate, thereby facilitating the proton transfer reactions. Moreover, the conformational and electrostatic changes associated with these reactions are not limited to the active site but rather extend throughout the entire enzyme. We also performed hybrid simulations for mutant KSIs that alter the hydrogen bonding in the active site.127 These calculations suggest that KSI forms a preorganized active site but that the structure of this preorganized active site is altered upon mutation. Moreover, small conformational changes due to stochastic thermal motions are required within this preorganized active site to facilitate the proton transfer reactions. In addition, we examined the hydrogen bonding interactions and NMR chemical shifts for a series of bound phenolates that have been studied experimentally by Dan Herschlag’s group to further understand the fundamental nature of the hydrogen bonding interactions.134 Our calculations indicated that the electronic inductive effects along a hydrogen bonding network of tyrosines strongly influence the hydrogen bonding interactions between the enzyme and substrate. We also investigated the structural and dynamical properties of water in the active site of wild-type KSI and several mutants.143
We have developed a theoretical formulation for proton-coupled electron transfer (PCET) reactions in enzymes. This theory includes the quantum mechanical effects of the active electrons and the transferring proton, as well as the motions of all atoms in the complete solvated enzyme system. We have derived a series of nonadiabatic rate constant expressions that are valid in well-defined regimes. In this theory, the rate constant and kinetic isotope effect (KIE) are strongly influenced by the equilibrium proton donor-acceptor distance and frequency, the vibronic coupling, the reaction free energy, and the protein/solvent reorganization energy
We have applied this theory to the PCET reaction catalyzed by SLO. This work was motivated by experiments from the Klinman group. Our calculations reproduced the experimentally observed magnitude and temperature dependence of the KIE without fitting any parameters directly to the experimental kinetic data. The large magnitude of the KIE of ~80 at room temperature arises mainly from the dominance of ground state tunneling and the relatively large ratio of overlaps for the hydrogen and deuterium vibrational wavefunctions. The weak temperature dependence of the KIE is due in part to the dominance of the local component of the proton donor-acceptor motion. We have also studied the impact of mutation of I553, which is ~15 Å from the active site iron. Our calculations indicate that the equilibrium proton transfer distance increases and the associated frequency decreases as residue 553 becomes less bulky, leading to the experimentally observed increase in the magnitude and temperature dependence of the KIE. The molecular dynamics simulations provide insight into how the effects of distal mutations are transmitted in enzymes to ultimately impact the catalytic rates. Currently we are performing docking calculations to examine the preferred orientation of the linoleic acid substrate.
This project is in collaboration with the Bevilacqua group at Penn State. We have used molecular dynamics simulations to study the long-distance communication between a distal structural portion of the ribozyme, namely the C41 base quartet, and the active site of the HDV ribozyme.129 We have also used molecular dynamics simulations to study the impact of C75 protonation on the structure and motions of the ribozyme and to examine the metal ion interactions with both a standard and a reverse GU wobble.137, 141 Protonation of C75 was observed to locally organize the active site in a manner that facilitates the catalytic mechanism in which C75+ acts as a general acid and Mg2+ as a Lewis acid. We identified two types of Mg2+ ions associated with the ribozyme, chelated and diffuse, at the reverse and standard GU wobbles, respectively, which appear to contribute to catalysis and stability, respectively. We also used quantum mechanical/molecular mechanical (QM/MM) methods to study the self-cleavage mechanism of the HDV ribozyme.150 Our calculations indicate that the self-cleavage reaction is concerted with a phosphorane-like transition state when a divalent ion, Mg2+ or Ca2+, is bound at the catalytic site but is sequential with a phosphorane intermediate when a monovalent ion, such as Na+, is at this site. These observations are consistent with available experimental data.