Direct Look at the Electric Field in Ketosteroid Isomerase and Its Variants

<div><p>Electrostatic interactions play a pivotal role in enzymatic catalysis and are increasingly modeled explicitly in computational enzyme design; nevertheless, they are challenging to measure experimentally. Using vibrational Stark effect (VSE) spectroscopy, we have measured electric fields inside the active site of the enzyme ketosteroid isomerase (KSI). These studies have shown that these fields can be unusually large, but it has been unclear to what extent they specifically stabilize the transition state (TS) relative to a ground state (GS). In the following, we use crystallography and computational modeling to show that KSI’s intrinsic electric field is nearly perfectly oriented to stabilize the geometry of its reaction’s TS. Moreover, we find that this electric field adjusts the orientation of its substrate in the ground state so that the substrate needs to only undergo minimal structural changes upon activation to its TS. This work provides evidence that the active site electric field in KSI is preorganized to facilitate catalysis and provides a template for how electrostatic preorganization can be measured in enzymatic systems. <br></p></div>

Extreme electric fields power catalysis in the active site of ketosteroid isomerase

Science ◽

10.1126/science.1259802 ◽

2014 ◽

Vol 346 (6216) ◽

pp. 1510-1514 ◽

Cited By ~ 215

Author(s):

Stephen D. Fried ◽

Sayan Bagchi ◽

Steven G. Boxer

Keyword(s):

Electric Field ◽

Electric Fields ◽

Active Site ◽

Stark Effect ◽

Catalytic Effect ◽

Biomolecular Systems ◽

Ketosteroid Isomerase ◽

Rate Determining Step ◽

Experimental Approaches ◽

A Charge

Enzymes use protein architecture to impose specific electrostatic fields onto their bound substrates, but the magnitude and catalytic effect of these electric fields have proven difficult to quantify with standard experimental approaches. Using vibrational Stark effect spectroscopy, we found that the active site of the enzyme ketosteroid isomerase (KSI) exerts an extremely large electric field onto the C=O chemical bond that undergoes a charge rearrangement in KSI’s rate-determining step. Moreover, we found that the magnitude of the electric field exerted by the active site strongly correlates with the enzyme’s catalytic rate enhancement, enabling us to quantify the fraction of the catalytic effect that is electrostatic in origin. The measurements described here may help explain the role of electrostatics in many other enzymes and biomolecular systems.

Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1111566109 ◽

2012 ◽

Vol 109 (6) ◽

pp. E299-E308 ◽

Cited By ~ 64

Author(s):

A. T. Fafarman ◽

P. A. Sigala ◽

J. P. Schwans ◽

T. D. Fenn ◽

D. Herschlag ◽

...

Keyword(s):

Electric Field ◽

Active Site ◽

Ketosteroid Isomerase ◽

Field Heterogeneity

A Preorganized Electric Field Leads to Minimal Geometrical Reorientation in the Catalytic Reaction of Ketosteroid Isomerase

Journal of the American Chemical Society ◽

10.1021/jacs.0c00383 ◽

2020 ◽

Vol 142 (22) ◽

pp. 9993-9998 ◽

Cited By ~ 4

Author(s):

Yufan Wu ◽

Stephen D. Fried ◽

Steven G. Boxer

Keyword(s):

Electric Field ◽

Catalytic Reaction ◽

Ketosteroid Isomerase

A Preorganized Electric Field Leads to Minimal Geometrical Reorientation in the Catalytic Reaction of Ketosteroid Isomerase

10.26434/chemrxiv.11559660.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Yufan Wu ◽

Stephen Fried ◽

Steven Boxer

Keyword(s):

Electric Field ◽

Electric Fields ◽

Ground State ◽

Active Site ◽

Electrostatic Interactions ◽

Stark Effect ◽

Structural Changes ◽

Enzymatic Catalysis ◽

Ketosteroid Isomerase ◽

Computational Enzyme Design

<div><p>Electrostatic interactions play a pivotal role in enzymatic catalysis and are increasingly modeled explicitly in computational enzyme design; nevertheless, they are challenging to measure experimentally. Using vibrational Stark effect (VSE) spectroscopy, we have measured electric fields inside the active site of the enzyme ketosteroid isomerase (KSI). These studies have shown that these fields can be unusually large, but it has been unclear to what extent they specifically stabilize the transition state (TS) relative to a ground state (GS). In the following, we use crystallography and computational modeling to show that KSI’s intrinsic electric field is nearly perfectly oriented to stabilize the geometry of its reaction’s TS. Moreover, we find that this electric field adjusts the orientation of its substrate in the ground state so that the substrate needs to only undergo minimal structural changes upon activation to its TS. This work provides evidence that the active site electric field in KSI is preorganized to facilitate catalysis and provides a template for how electrostatic preorganization can be measured in enzymatic systems. <br></p></div>

An Ab Initio QM/MM Study of the Electrostatic Contribution to Catalysis in the Active Site of Ketosteroid Isomerase

Molecules ◽

10.3390/molecules23102410 ◽

2018 ◽

Vol 23 (10) ◽

pp. 2410 ◽

Cited By ~ 3

Author(s):

Xianwei Wang ◽

Xiao He

Keyword(s):

Electric Field ◽

Ab Initio ◽

Active Site ◽

Md Simulation ◽

Stark Effect ◽

Computational Simulation ◽

Ketosteroid Isomerase ◽

Correlation Relationship ◽

Amber Force Field ◽

Electrostatic Contribution

The electric field in the hydrogen-bond network of the active site of ketosteroid isomerase (KSI) has been experimentally measured using vibrational Stark effect (VSE) spectroscopy, and utilized to study the electrostatic contribution to catalysis. A large gap was found in the electric field between the computational simulation based on the Amber force field and the experimental measurement. In this work, quantum mechanical (QM) calculations of the electric field were performed using an ab initio QM/MM molecular dynamics (MD) simulation and electrostatically embedded generalized molecular fractionation with conjugate caps (EE-GMFCC) method. Our results demonstrate that the QM-derived electric field based on the snapshots from QM/MM MD simulation could give quantitative agreement with the experiment. The accurate calculation of the electric field inside the protein requires both the rigorous sampling of configurations, and a QM description of the electrostatic field. Based on the direct QM calculation of the electric field, we theoretically confirmed that there is a linear correlation relationship between the activation free energy and the electric field in the active site of wild-type KSI and its mutants (namely, D103N, Y16S, and D103L). Our study presents a computational protocol for the accurate simulation of the electric field in the active site of the protein, and provides a theoretical foundation that supports the link between electric fields and enzyme catalysis.

Resolution in photoelectron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100086593 ◽

1991 ◽

Vol 49 ◽

pp. 458-459

Author(s):

G. F. Rempfer

Keyword(s):

Electron Microscopy ◽

Electric Field ◽

Ultraviolet Light ◽

Uniform Field ◽

Virtual Image ◽

Lens System ◽

Photoemission Electron Microscopy ◽

Planar Electrode ◽

Electron Lens ◽

Photoemission Electron

In photoelectron microscopy (PEM), also called photoemission electron microscopy (PEEM), the image is formed by electrons which have been liberated from the specimen by ultraviolet light. The electrons are accelerated by an electric field before being imaged by an electron lens system. The specimen is supported on a planar electrode (or the electrode itself may be the specimen), and the accelerating field is applied between the specimen, which serves as the cathode, and an anode. The accelerating field is essentially uniform except for microfields near the surface of the specimen and a diverging field near the anode aperture. The uniform field forms a virtual image of the specimen (virtual specimen) at unit lateral magnification, approximately twice as far from the anode as is the specimen. The diverging field at the anode aperture in turn forms a virtual image of the virtual specimen at magnification 2/3, at a distance from the anode of 4/3 the specimen distance. This demagnified virtual image is the object for the objective stage of the lens system.

Field-assisted ionization theory for microscopists

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100171833 ◽

1994 ◽

Vol 52 ◽

pp. 820-821

Author(s):

Patrick P. Camus

Keyword(s):

Electric Field ◽

Electron Tunneling ◽

Ionization Probability ◽

Critical Distance ◽

Tunneling Probability ◽

Field Ionization ◽

Radius Of Curvature ◽

Field Evaporation ◽

A Value ◽

Ion Emission

The theory of field ion emission is the study of electron tunneling probability enhanced by the application of a high electric field. At subnanometer distances and kilovolt potentials, the probability of tunneling of electrons increases markedly. Field ionization of gas atoms produce atomic resolution images of the surface of the specimen, while field evaporation of surface atoms sections the specimen. Details of emission theory may be found in monographs.Field ionization (FI) is the phenomena whereby an electric field assists in the ionization of gas atoms via tunneling. The tunneling probability is a maximum at a critical distance above the surface,xc, Fig. 1. Energy is required to ionize the gas atom at xc, I, but at a value reduced by the appliedelectric field, xcFe, while energy is recovered by placing the electron in the specimen, φ. The highest ionization probability occurs for those regions on the specimen that have the highest local electric field. Those atoms which protrude from the average surfacehave the smallest radius of curvature, the highest field and therefore produce the highest ionizationprobability and brightest spots on the imaging screen, Fig. 2. This technique is called field ion microscopy (FIM).

Electric field

AccessScience ◽

10.1036/1097-8542.216000 ◽

2015 ◽

Keyword(s):

Electric Field

Simple quasi-two-dimensional analytical model to characterise the electric field in an LDD MOSFET

IEE Proceedings G Circuits Devices and Systems ◽

10.1049/ip-g-2.1990.0044 ◽

1990 ◽

Vol 137 (4) ◽

pp. 291

Author(s):

R.H. Patel ◽

D.-L. Kwong ◽

N. Herr

Keyword(s):

Electric Field ◽

Analytical Model ◽

Two Dimensional