Simulations of remote mutants of dihydrofolate reductase reveal the nature of a network of residues coupled to hydride transfer

Abstract

Recent experimental and theoretical studies have proposed that enzymes involve networks of coupled residues throughout the protein that participate in motions accompanying chemical barrier crossing. Here, we have examined portions of a proposed network in dihydrofolate reductase (DHFR) using quantum mechanics/molecular mechanics simulations. The simulations use a hybrid quantum mechanics-molecular mechanics approach with a recently developed semiempirical AM1-SRP Hamiltonian that provides accurate results for this reaction. The simulations reproduce experimentally determined catalytic rates for the wild type and distant mutants of E. coli DHFR, underscoring the accuracy of the simulation protocol. Additionally, the simulations provide detailed insight into how residues remote from the active site affect the catalyzed chemistry, through changes in the thermally averaged properties along the reaction coordinate. The mutations do not greatly affect the structure of the transition state near the bond activation, but we observe differences somewhat removed from the point of C-H cleavage that affect the rate. The mutations have global effects on the thermally averaged structure that propagate throughout the enzyme and the current simulations highlight several interactions that appear to be particularly important.

Keywords: AM1-SRP; QM/MM; enzyme dynamics; hydrogen tunneling; network of interactions.

Figure 1

QM/MM partitioning scheme. The dashed line divides the QM and MM regions, and the quantum hydrogen link atoms are circled. Reproduced from ref. 25 with permission from ACS.

Figure 2

Classical mechanical PMFs for ecDHFR and a series of mutants. Each PMF represents 200 ps of sampling in each of 13-15 windows along the reaction coordinate. Thermodynamic parameters from these PMFs are listed in Table 1.

Figure 3

Distance matrices showing changes in distance between α-carbons of each enzyme in going from the reactant to the transition states. The axes are residue numbers and the color at each point indicates the change in distance between each pair of residues in going from the reactant to the TS. The color scale follows the visible spectrum with blue corresponding to -3 Å (residues are closer at TS than reactant) and red corresponding to +3 Å (residues are farther apart at TS than reactant). ABD=Adenosine Binding Domain. We note that these matrices are symmetric across the diagonal (y=x).

Scheme 1

The hydride transfer catalyzed by DHFR.