Designed Local Electric Fields-Promising Tools for Enzyme Engineering

Abstract

Designing efficient catalysts is one of the ultimate goals of chemists. In this Perspective, we discuss how local electric fields (LEFs) can be exploited to improve the catalytic performance of supramolecular catalysts, such as enzymes. More specifically, this Perspective starts by laying out the fundamentals of how local electric fields affect chemical reactivity and review the computational tools available to study electric fields in various settings. Subsequently, the advances made so far in optimizing enzymatic electric fields through targeted mutations are discussed critically and concisely. The Perspective ends with an outlook on some anticipated evolutions of the field in the near future. Among others, we offer some pointers on how the recent data science/machine learning revolution, engulfing all science disciplines, could potentially provide robust and principled tools to facilitate rapid inference of electric field effects, as well as the translation between optimal electrostatic environments and corresponding chemical modifications.

Figure 1

(a) Generic Diels–Alder reaction with x-, y-, z-axes defined. The direction of the dipole moment and the electric field direction that will result in catalysis are shown in the Gaussian convention. (b) Enantiomeric transition state structures for another Diels–Alder reaction. The individual structures can be selectively stabilized with the help of an EEF oriented along the x-axis (positive and negative signs indicate the respective stabilizing field direction). (c) Exo/endo transition states for a similar Diels–Alder reaction. Here, and EEF oriented along the y-axis controls the relative stability. (d) Endo/exo as well as R and S enantiomeric transition state complexes for a final example Diels–Alder reaction. Here, positive and negative signs show the directions of the x and y-aligned electric fields that result in selective stabilization. Adapted with permission from ref (15). Copyright 2020 American Chemical Society. (e) Generally, reaction rates increase in one direction of EEF and decrease in another. However, as the magnitude of the EEF rises, the inhibitory direction changes to a rate-enhancing one after the highest inhibition point has been reached. Adapted with permission from ref (10). Copyright 2023 Wiley.

Figure 2

(a) Schematic representation of the Claisen rearrangement of chorismate to prephenate through a chairlike TS. (b) Kinetic parameters for WT chorismate mutase and its three variants. Red and green indicate loss and gain in the reactivity of enzyme, respectively. (c) Reaction axis (C–O bond axis) overlaid on the TS structure, and local electric field quantification in the WT enzyme along the reaction axis.

Figure 3

(a) Quantified net dipole moment and enzyme’s LEF along the Fe–O axis of the reactant and intermediate in CYP450_BSβ. (b) Quantified net dipole moment and enzyme’s LEF along the Fe–O axis of the reactant and intermediate in CYP450_OleT. (c) Cartesian axis and the Gaussian convention used for electric field and dipole moment vectors throughout this perspective. (d) Electrostatic stabilization energy in kcal/mol obtained from interaction of the LEFs with the dipole moment of the substrate. Note that RC, TS, and IM stand for reactant, transition state, and intermediate, respectively.

Figure 4

Average local electric field (LEF) gradually attenuates along the Fe–O axis within different categories of heme-iron proteins, specifically mono-oxygenase, peroxidase, and catalase (from left to right). This observed trend is complemented by the illustration of the electric field convention employed in the study, showcased on the right side of the figure. Note that 1 MV cm^–1 = 10^–2 V Å^–1. For the electric field vector, the physics convention was used.

Figure 5

Promotion and inhibition of the state I and II (active oxidant Fe=O formation and substrate oxidation) by IEF, respectively.

Figure 6

Reaction and pivotal electrostatic interactions within the active site of TEM β-lactamases. (a) Illustration of the PenG acylation process. The hydroxyl group of S70 functions as a nucleophile, engaging the β-lactam C=O bond of PenG. This leads to the creation of an oxyanion intermediate, which subsequently transforms into an acyl-enzyme complex. (b) Main three mutants, WT, A237Y, and A237Y^e, each accompanied by its corresponding electric field vector, F, along the C=O reaction axis. The magnitudes of the electric fields are indicated for all three mutants along with their respective acylation rates. Herein, note that 1 MV cm^–1 = 10^–2 V Å^–1.

Figure 7

(a) Outlined are the conjectured catalytic processes underpinning the function of PaAPase. These proposed mechanisms delve into the intricate steps and interactions that likely drive the enzyme’s catalytic activity: His171-phosphorylation, AsA-phosphorylation, and phospho-His171 hydrolysis. (b) Catalytic residues, the reaction axis in the active site and employing directed evolution techniques to refine the performance of the original enzyme Q0 (PaAPase) with the aim of augmenting its ability to phosphorylate AsA along with their r_t/r_h ratio and percentage conversion. (c) N–P–O negative reaction axis. Direction of positive and negative LEF for achieving phosphorylation vs hydrolysis, respectively. And the LEF exerted by WT and Q6. Figure (b) is adapted with permission from ref (72). Copyright 2021 American Chemical Society.

Figure 8

(a) Operational mechanism underlying aldehyde hydrogenation, facilitated by LADH and utilizing NADH as the cofactor. RS and TS stand for reactant state and transition state, respectively. Electric field and dipole moment directions are shown by F_enz and μ vectors. (b) The electric field F_enz at the active site reduces the activation energy barrier by ΔΔG^‡, achieved by selectively stabilizing the transition state (μ_TS) over the reactant state (μ_RS). It is essential to recognize that these fields result from charges and dipoles arranged within the protein structure, distinct from externally applied electric fields. (c) The different variants of the WT enzyme created: WT, Ser48Thr, Zn²⁺ → Co²⁺, and Co,S48T. Their enzymatic LEF is also shown at the bottom of each variant.

Scheme 1. Kemp Elimination Reaction, Involving a Singular Proton Transfer from the 5-Nitrobenzisoxazole Substrate by a Catalytic Base Leading to the Rupture of the 5-Membered Ring and the Formation of the End Product, α-Cyanophenol

F vectors showing the projection of the Enzymatic LEFs.

Figure 9

Further modifications done in the model cage to achieve absolute R/S-selective porphyrin boxes: mod1 and mod2. The y-LEF is also shown for each cage. The substitutions introduced are present at the bottom right.