Probing the Electrostatics of Active Site Microenvironments along the Catalytic Cycle for Escherichia coli Dihydrofolate Reductase

Abstract

Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and 13C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor–acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for significant electrostatic changes in the active site microenvironments due to conformational motion occurring over the catalytic cycle of ecDHFR.

Site-Specific Infrared Probes of Proteins

“…Specifically, by analyzing the lineshape of the C≡N stretching vibration as well as the chemical shift of the nitrile moiety placed in the protein's binding pocket, they were able to develop a model to quantitatively determine the protonation states of the ligand and tyrosine residues in the enzyme active site in a site-specific manner, and account for how the protonation states of these moieties change as a function of the charge density of the ligand. In a similar application, Benkovic and coworkers (87) employed SCN to study the role of electrostatics at each stage of the catalytic cycle of dihydrofolate reductase (DHFR). Through a combination of IR and NMR measurements of the SCN probe and with the help of MD simulations and theoretical calculations, they were able to determine the changes in the structure and local electric field magnitude of the enzyme active site along the protein’s catalytic cycle.…”

Protein motions and dynamic effects in enzyme catalysis

“…39 Progression through the catalytic cycle has also been studied through the use of infrared probes. [51][52][53] Hydride transfer is simply not possible in the occluded conformation as the reactants are not sufficiently close to one another. More generally, the ability to form the occluded conformation does not affect the chemical step of the catalytic cycle directly, as shown both by the similarity of the single turnover rate constants of EcDHFR and MpDHFR at pH 7 12,48 and by the existence of EcDHFR variants such as EcDHFR-S148A that are incapable of forming an occluded conformation yet maintain wild-type-like single turnover rate constants.…”

Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers: Temporal order and the roles of Asp27 and Tyr100

“…The direct source of the proton is likely to be a water molecule that could enter the active site as the Met20 loop changes conformations (18), and crystal data supporting this hypothesis are provided in the accompanying work by Wan et al (32). Y100 has been implicated to provide electrostatic stabilization of the developing positive charge on the nicotinamide moiety, as well as exhibiting strong electrostatic interactions with NADP + and folate (20,21).…”

“…3.5 Å from both the amide of the nicotinamide and N8 of the pterin ring ( Fig. 1), has been shown to play an important role in electrostatically facilitating the ecDHFR-catalyzed reaction (20,21). The location of Y100 suggests that it may form hydrogen bonds with both ligands to control the distance between them.…”