Electrostatic interactions help an enzyme do its job

Marian Szebenyi, MacCHESS, Cornell University

A catalytic enzyme facilitates a reaction by bringing one or more molecules into its active site and there providing an environment conducive to the reaction.

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Figure 1. A, ecDHFR in the closed conformation with folate (FOL) and NADP+ bound. Modified residues in L45C-CN and T46C-CN mutants are shown. B, Closed (red) and occluded (blue) conformations of ecDHFR. C, Major complexes in the catalytic cycle of ecDHFR, color-coded according to ecDHFR conformation at each stage.

Electrostatic interactions are important for both phases, and conformational changes occurring in an enzyme during its catalytic cycle modify these interactions. For complete understanding of the catalytic process, we require knowledge of the contribution of electrostatic effects to each step in the process, as well as an understanding of how conformational changes affect the electrostatic environment in the active site.

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent conversion of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF), a step in the synthesis of some amino acids. During the catalytic process, the NADP+/NADPH cofactor is bound, altered, and released, as is the DHF/THF substrate, and the enzyme conformation switches between “closed” and “occluded” forms (Figure 1C). Using a combination of techniques, the Benkovic group (Penn. State) has followed the electrostatic microenvironment in the active site of E. coli DHFR (ecDHFR) throughout its catalytic cycle. As aids to monitoring the state of the active site, two mutant DHFRs were generated, each introducing a new Cys residue which could be modified by addition of a -CN (sometimes -13CN) reporter group (Figure 1A).

The CN vibrational stretching frequency, measured by FTIR (Fourier transform infrared) spectroscopy, and the 13C chemical shift, measured using NMR (nuclear magnetic resonance), were sensitive to the electrostatic environment of the modified residue. To determine the exact location of the reporter groups, crystal structures were determined for the two mutants, to about 2 Å resolution, using data taken at CHESS A1 station. Structures were determined using molecular replacement, with the wild-type structure as a model: the backbones for the mutants were nearly the same as for the wild-type, with an RMSD of the heavy atoms of just 0.5 Å. Kinetics measurements verified that the activity of the mutant enzymes was comparable to that of the wild-type.

FTIR and NMR measurements, along with QM/MM (quantum mechanics/molecular dynamics) simulations, provided information about the electrostatics, as well as the degree of hydration, of the environment of the CN probes. Moreover, it was possible to calculate the contributions of the ligands, surrounding residues, and solvent molecules. Significant changes occurred as the enzyme progressed along the catalytic cycle, particularly near the hydride transfer site, with smaller changes in the folate-binding pocket. The interpretation of these changes is that electrostatic interactions between protein and ligands act to orient the reactants in such a way as to create an electric field favoring the hydride transfer reaction; active site residues also contribute to this field. Future work with more probes and additional states of DHFR will expand knowledge of electrostatics in DHFR to other parts of the molecule and other conformational states.

References:

[1] C.T. Liu, J.P. Layfield, R.J. Stewart III, J.B. French, P. Hanoian, J.B. Asbury, S. Hammes-Schiffer, S.J. Benkovic, "Probing the electrostatics of active site microenvironments along the catalytic cycle for Escherichia coli dihydrofolate reductase," J. Am. Chem. Soc. 136, 10349-10360 (2014).