Electrostatics in Enzyme Catalysis

About the Figure: Using a well-calibrated vibrational probe, many diverse intermolecular interactions can be quantified in the unifying physical metric of electric fields including both general and specific interactions, e.g. H-bonds.

The Boxer Lab has established and utilized the Stark effect and Stark spectroscopy to quantify the electric fields that characterize non-covalent intermolecular interactions, as shown in the figure to the left. With long-standing work on electronic Stark spectroscopy, we pioneered vibrational Stark spectroscopy (VSS) [122], which allows for a quantitative model with which to interpret IR frequency shifts as changes in a physical force, the electric field of an environment acting upon a bond [176]. 

Using this framework, the Boxer lab has used the vibrational Stark effect (VSE) to quantify functionally relevant electric fields in proteins, for example in the active site of the enzyme Ketosteroid Isomerase (KSI) [303]. Through measurements of the activation free energy barrier (from transition state theory) and the electric field experienced at a functionally relevant C=O on a TS-like inhibitor, the contribution of electric fields to the catalytic proficiency of KSI has been measured for the first time. Subsequent studies, using Amber-suppression, via introducing unnatural Cl-Y-derivatives, showed that both subtle and perturbative mutations can be explained using the VSE [310]. Further work in the lab has demonstrated that C=O probes are well-characterized and functionally relevant probes for studying many enzymatic systems [316]. Current work expands on the scope of how electric fields are connected with enzymatic function and enzyme evolution, enzyme design, solvation, photoisomerization, and non-biological catalysis. 

About the Figure: (Top) The general mechanism for the isomerization reaction catalyzed by KSI, whereby the functionally relevant carbonyl undergoes significant charge rearrangement over the course of the reaction. (Bottom left) The electric field-frequency calibration curve for a C=O probe in both organic and aqueous solvents, which provides a mapping between an observed IR frequency and an electric field. (Bottom middle) The general model for electrostatic catalysis whereby an electric field can only have a beneficiary effect on catalysis through preferential stabilization of the transition state's (TS) dipole relative to the ground state (GS). (Bottom right) A quantification of the electric field contribution to KSI's catalytic rate using both traditional mutagenesis and non-canonical amino acid incorporation, whereby a linear relationship is observed between the free energy barrier and the active site electric field. The slope and intercept of the best-fit line relate to the extent of charge transfer between the TS and GS and the activation barrier in the absence of an electric field, respectively, the latter providing the electrostatic contribution to catalysis. 

[316]  "Electric Fields and Enzyme Catalysis", Stephen D. Fried and Steven G. Boxer, Annual Reviews of Biochemistry,86, 387-415 (2017). [pdf]

[315]  "Solvent-Independent Anharmonicity for Carbonyl Oscillators", Samuel H. Schneider, Huong T. Kratochvil, Martin T. Zanni, and Steven G. Boxer, J. Phys. Chem. B, 121, 2331−2338 (2017). [pdf]

[311]  "Vibrational Stark Effects of Carbonyl Probes Applied to Re-interpret IR and Raman Data for Enzyme Inhibitors in Terms of Electric Fields at the Active Site", Samuel Hayes Schneider, and Steven G. Boxer, J. Phys. Chem. B, 120, 9672-9684 (2016). [pdf]


[310]  "A Critical Test of the Electrostatic Contribution to Catalysis with Non-canonical Amino Acids in Ketosteroid Isomerase", Yufan Wu, and Steven G. Boxer, J. Am. Chem. Soc., 138, 11890-11895 (2016). [pdf]

[303]  “Extreme Electric Fields Power Catalysis in the Active Site of Ketosteroid Isomerase”, Stephen D. Fried, Sayan Bagchi, Steven G. Boxer, Science, 346, 1510-1514 (2014). [pdf] [Perspective]

The Boxer Laboratory, Stanford University, Department of Chemistry, Stanford, CA, 94305-5012

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