Effect of Asp122 Mutations on E. Coli DHFR: Structure and Dynamics

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) in the presence of NADPH as a hydride donor. The key steps in the reaction are accomplished by correlated protein motions in the Michaelis-Menten (E:NADPH:DHF) complex. The present theoretical study employs mutagenesis to examine the relation between structural and functional properties of the enzyme. We mutate Asp122 in Escherichia coli DHFR, which is a highly conserved amino acid in the DHFR family.¹ The consequent effect of the mutation on the enzyme catalysis is examined both structurally and energetically. Furthermore, we inspected the ribose sugar conformational behavior, which is expected to influence ligand binding and hydride donor–acceptor dynamics during the chemical reaction. We employed a hybrid quantum-mechanical/molecular mechanical (QM/MM) approach with a recently developed semi-empirical AM1-SRP Hamiltonian that provides accurate results for this reaction.^2-5 Our investigations reveal that the structural and dynamic perturbations caused by Asp122 mutations are along the reaction coordinate and lower the rate of hydride transfer. Altered coupled motions in the enzyme suggest that the possible dynamic coupling is lost as a result of the mutation. These effects are also reflected in the ribose sugar puckering profile which unveils that the fine balance of interactions is perturbed by the mutations. The role of water during the catalysis is also investigated suggesting networks of hydrogen bonded water molecules near the active site.

References:

1. Agarwal, P. K.; Billeter, S. R.; Rajagopalan, P. T. R.; Benkovic, S. J.; Hammes-Schiffer, S., Network of coupled promoting motions in enzyme catalysis. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (5), 2794-2799.
2. Warshel, A.; Levitt, M., Theoretical studies of enzymic reactions - dielectric, electrostatic and steric stabilization of carbonium-ion in reaction of lysozyme. J. Mol. Biol. 1976, 103 (2), 227-249.
3. Dewar, M. J. S., Applications of quantum-mechanical molecular-models to chemical problems .70. Quantum-mechanical molecular-models. J. Phys. Chem. 1985, 89 (11), 2145-2150.
4. Rossi, I.; Truhlar, D. G., Parameterization of NDDO wavefunctions using genetic algorithms. An evolutionary approach to parameterizing potential energy surfaces and direct dynamics calculations for organic reactions. Chem. Phys. Lett. 1995, 233, 231-236.
5. Doron, D.; Major, D. T.; Kohen, A.; Thiel, W.; Wu, X., Hybrid Quantum and Classical Simulations of the Dihydrofolate Reductase Catalyzed Hydride Transfer Reaction on an Accurate Semi-Empirical Potential Energy Surface. J. Chem. Theory Comput. 2011, 7 (10), 3420-3437.