Perspectives on electrostatics and conformational motions in enzyme catalysis

Abstract

CONSPECTUS: Enzymes are essential for all living organisms, and their effectiveness as chemical catalysts has driven more than a half century of research seeking to understand the enormous rate enhancements they provide. Nevertheless, a complete understanding of the factors that govern the rate enhancements and selectivities of enzymes remains elusive, due to the extraordinary complexity and cooperativity that are the hallmarks of these biomolecules. We have used a combination of site-directed mutagenesis, pre-steady-state kinetics, X-ray crystallography, nuclear magnetic resonance (NMR), vibrational and fluorescence spectroscopies, resonance energy transfer, and computer simulations to study the implications of conformational motions and electrostatic interactions on enzyme catalysis in the enzyme dihydrofolate reductase (DHFR). We have demonstrated that modest equilibrium conformational changes are functionally related to the hydride transfer reaction. Results obtained for mutant DHFRs illustrated that reductions in hydride transfer rates are correlated with altered conformational motions, and analysis of the evolutionary history of DHFR indicated that mutations appear to have occurred to preserve both the hydride transfer rate and the associated conformational changes. More recent results suggested that differences in local electrostatic environments contribute to finely tuning the substrate pKa in the initial protonation step. Using a combination of primary and solvent kinetic isotope effects, we demonstrated that the reaction mechanism is consistent across a broad pH range, and computer simulations suggested that deprotonation of the active site Tyr100 may play a crucial role in substrate protonation at high pH. Site-specific incorporation of vibrational thiocyanate probes into the ecDHFR active site provided an experimental tool for interrogating these microenvironments and for investigating changes in electrostatics along the DHFR catalytic cycle. Complementary molecular dynamics simulations in conjunction with mixed quantum mechanical/molecular mechanical calculations accurately reproduced the vibrational frequency shifts in these probes and provided atomic-level insight into the residues influencing these changes. Our findings indicate that conformational and electrostatic changes are intimately related and functionally essential. This approach can be readily extended to the study of other enzyme systems to identify more general trends in the relationship between conformational fluctuations and electrostatic interactions. These results are relevant to researchers seeking to design novel enzymes as well as those seeking to develop therapeutic agents that function as enzyme inhibitors.

Figure 1

Catalytic mechanism of ecDHFR involving proton transfer from an active site water molecule to N5 of DHF (blue) and subsequent hydride transfer from NADPH to C6 of DHF (red).

Figure 2

(A) Ribbon structure of ecDHFR with bound NADP⁺ (green) and folate (sky blue) illustrating the differences between the closed (PDB ID 3QL3; red) and occluded (PDB ID 1RX6; blue) conformations of the Met20 loop. (B) The ecDHFR catalytic cycle distinguishing the closed conformation (red) and occluded conformation (blue). Adapted with permission from ref (10). Copyright 2014 American Chemical Society.

Figure 3

Conformational changes measured by thermally averaged C_α–C_α distance changes from the reactant state to the transition state of the hydride transfer reaction observed in EVB-MD simulations of WT ecDHFR, N23PP ecDHFR, and N23PP/G51PEKN ecDHFR. For clarity, only distances that increase are shown on the left side of each plot, while only distances that decrease are shown on the right.

Figure 4

Thiocyanate probes in the ecDHFR active site colored according to their FTIR maximum vibrational frequency (cm^–1) for closed (red) and occluded (blue) states along the catalytic cycle. When present, folate or THF is shown in sky blue and NADP(H) is shown in green.

Figure 5

Ribbon structure of ecDHFR colored according to residue-based contributions to the calculated electric field along the hydride transfer D–A axis (depicted as an orange arrow) for selected residues from MD simulations of the model Michaelis complex E:FOL:NADP⁺. Positive values of the electric field disfavor hydride transfer, while negative values facilitate hydride transfer. NADP⁺ is colored green, and folate is colored sky blue.