Modulating Enzyme Activity by Altering Protein Dynamics with Solvent

Abstract

Optimal enzyme activity depends on a number of factors, including structure and dynamics. The role of enzyme structure is well recognized; however, the linkage between protein dynamics and enzyme activity has given rise to a contentious debate. We have developed an approach that uses an aqueous mixture of organic solvent to control the functionally relevant enzyme dynamics (without changing the structure), which in turn modulates the enzyme activity. Using this approach, we predicted that the hydride transfer reaction catalyzed by the enzyme dihydrofolate reductase (DHFR) from Escherichia coli in aqueous mixtures of isopropanol (IPA) with water will decrease by ∼3 fold at 20% (v/v) IPA concentration. Stopped-flow kinetic measurements find that the pH-independent k_hydride rate decreases by 2.2 fold. X-ray crystallographic enzyme structures show no noticeable differences, while computational studies indicate that the transition state and electrostatic effects were identical for water and mixed solvent conditions; quasi-elastic neutron scattering studies show that the dynamical enzyme motions are suppressed. Our approach provides a unique avenue to modulating enzyme activity through changes in enzyme dynamics. Further it provides vital insights that show the altered motions of DHFR cause significant changes in the enzyme's ability to access its functionally relevant conformational substates, explaining the decreased k_hydride rate. This approach has important implications for obtaining fundamental insights into the role of rate-limiting dynamics in catalysis and as well as for enzyme engineering.

Figure 1:. Impact of isopropanol-water mixture on the transition state for hydride transfer.

(A) The averaged transition state structure calculated based on EVB computer simulations indicates no difference in the active-site residues (Phe31 and Tyr100 are shown) as well as cofactor (NADPH) and substrate (protonated DHF). 1,000 structures within ± 1kcal/mol (collective reaction coordinate) of the activation energy barrier were used to calculate the average structure. The transferring hydride is shown as a sphere. (B) The averaged electrostatic potential (+5kT/e in blue and −5kT/e per electron in red) for the transition state structures is practically the same (obtained by averaging the APBS calculated electrostatic potential of 1,000 TS structures). The only minor difference between the 0% and 20% IPA cases occurs around residues 98–99 (marked by small black arrow).

Figure 2:. IPA alters the functionally relevant conformational sub-states for catalysis.

Computer simulations with the EVB method were used to model the rate-limiting hydride transfer reaction in water (0% IPA), 20% IPA and 25% IPA. (The color coding is based on the reaction coordinate. See the free energy profiles in supporting information for details.) The 3-D plot (left panel) shows three different independent component vectors from QAA and the ellipses identify the conformational sub-states for each of the simulations. Each dot represents a single enzyme conformation along the reaction pathway of hydride transfer (color coded according to the value of the reaction coordinate). For each of the simulations, we identified conformational sub-states close to TS area (cyan-green-yellow dots). The mixed sub-state (M) represents a mixture of all other sub-states. The conformational fluctuations that enable the sampling of these sub-states are indicated by arrows (M ⟶I, M ⟶II, and M⟶III). In the middle panel, the structural changes in DHFR associated with fluctuations are shown in a movie like fashion, with regions displaying the largest fluctuations shown in color (Note these colors have no connection to reaction coordinate colors but are used to indicate same regions of protein that show motion). In these conformational fluctuations, the enzyme regions including Met20 and βF-βG loops show the largest displacements. The right-most panel indicates the activation of enzyme (visit into the TS conformational sub-states) by these protein motions. These activation profile corresponds to the 3 QAA modes shown on left hand side and are calculated by projecting the sampled conformational snapshots on each mode. Note that the conformational sub-states are clearly defined for 0% IPA; however, the enzyme landscape is altered significantly by addition of IPA in solution. At 20% and 25% these sub-states are not clearly defined, which indicate difficulty in sampling the motions (see the activation profiles in right panel) and reaching the functionally relevant sub-states in the TS area.

Figure 3:. Effect of isopropanol-water mixture on DHFR catalyzed hydride transfer.

Stop flow kinetics indicates that the pH independent rate for hydride transfer is 1170 ± 10 ^s−1 (black curve) for 0% isopropanol (buffer only), while it is 530 ± 10 ^s−1 with 20% isopropanol.

Figure 4:. Effect of isopropanol-water mixture on DHFR structure.

The X-ray structure indicates little difference in the electron density (left) and fitted structure (right) for the two cases. The gray cartoon shows the 0% IPA structure for orientation purposes only.

Figure 5:. Effect of IPA on DHFR dynamics.

(A) Background subtracted quasi-elastic neutron scattering (QENS) data from DHFR hydrogen atoms in solution at the indicated IPA concentration. The points show experimental data and line indicates fit to the model as described in supporting information. (B) The contribution to the QENS signal comes from hydrogen local motions and the translational and center-of-mass rotational movement of the entire enzyme in solution. A random path is illustrated by the black line where the center-of-mass translational and rotational motion of DHFR will be affected by the viscosity of the solution. The local dynamics of hydrogens (D_INT) attached to the side chain were modeled as diffusion in a sphere. (C) The internal motion diffusion coefficient, D_INT, at the indicated IPA solvent concentrations. The plot shows the sensitivity of D_INT at fixed values of the radius defining the extent of internal motion and the vibrational, oscillation Debye-Waller factor. As IPA concentration is initially increased to 2 0 %, D_INT decreases by approximately 60%. There is little difference between the 2 0 % and 30% samples. (D) Dependence of viscosity (η), D_INT, and pH independent rate for hydride transfer (k_{hydride,pH ‒ independent}) with isopropanol concentration. The first two quantities are for deuterated solutions while the last is from protonated solutions. Note, the uncertainties associated with the viscosity measurements are less than the size of the symbols. D_INT is from Table S3.

Figure 6:. Differential interaction of IPA with DHFR surface residues of DHFR.

100ns molecular dynamics trajectories were used to calculate relative occupancies of IPA molecules close to the surface of DHFR (see supporting information for more details). Regions of low interaction are shown in blue and higher occupancy in red. The cofactor and substrate are shown in magenta. The dynamical regions Met20 and βF-βG loops show little preference for IPA (and therefore more preference for water). Note, these results are based on difference between simulations with 15% and 30% IPA, other results were qualitatively similar.