ArticleProceedings of the National Academy of Sciences of the United States of America2012
Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 45 papers.
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Who cites it
45 citing papers in PubMed.
- Engineering drug-protein kinetics via intrinsic quantum Stark fields in binding motifs.Journal of molecular modeling · 2025Article
- Electric Field-Induced Sequential Prototropic Tautomerism in Enzyme-like Nanopocket Created by Single Molecular Break Junction.Journal of the American Chemical Society · 2024Article
- Electric Fields in Polymeric Systems.Chemical reviews · 2024Review
- Good Vibrations Report on the DNA Quadruplex Binding of an Excited State Amplified Ruthenium Polypyridyl IR Probe.Journal of the American Chemical Society · 2023Article
- Automated Variable Electric-Field DFT Application for Evaluation of Optimally Oriented Electric Fields on Chemical Reactivity.The Journal of organic chemistry · 2023Article
- Proton transfer reactions: From photochemistry to biochemistry and bioenergetics.BBA advances · 2023Article
- Nitrile Infrared Intensities Characterize Electric Fields and Hydrogen Bonding in Protic, Aprotic, and Protein Environments.Journal of the American Chemical Society · 2022Article
- Protein Electrostatics Investigated through Paramagnetic NMR for Nonpolar Groups.The journal of physical chemistry. B · 2022Article
- Interfacial Acid-Base Equilibria and Electric Fields Concurrently Probed byJournal of the American Chemical Society · 2021Article
- De novo determination of near-surface electrostatic potentials by NMR.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
- Electric Field Measurements Reveal the Pivotal Role of Cofactor-Substrate Interaction in Dihydrofolate Reductase Catalysis.ACS catalysis · 2020Article
- A Preorganized Electric Field Leads to Minimal Geometrical Reorientation in the Catalytic Reaction of Ketosteroid Isomerase.Journal of the American Chemical Society · 2020Article
- Article
- Cyanylated Cysteine Reports Site-Specific Changes at Protein-Protein-Binding Interfaces Without Perturbation.Biochemistry · 2018Article
- Electric Fields and Enzyme Catalysis.Annual review of biochemistry · 2017Review
- Minimalist IR and fluorescence probes of protein function.Current opinion in chemical biology · 2016Review
- Mechanism of endonuclease cleavage by the HigB toxin.Nucleic acids research · 2016Article
- Vibrational Stark Effects of Carbonyl Probes Applied to Reinterpret IR and Raman Data for Enzyme Inhibitors in Terms of Electric Fields at the Active Site.The journal of physical chemistry. B · 2016Article
- Dissecting Proton Delocalization in an Enzyme's Hydrogen Bond Network with Unnatural Amino Acids.Biochemistry · 2015Article
- Site-specific infrared probes of proteins.Annual review of physical chemistry · 2015Review
Corrections and comments
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Authors and funding
6 authors.
Funding
Abstract
Understanding the electrostatic forces and features within highly heterogeneous, anisotropic, and chemically complex enzyme active sites and their connection to biological catalysis remains a longstanding challenge, in part due to the paucity of incisive experimental probes of electrostatic properties within proteins. To quantitatively assess the landscape of electrostatic fields at discrete locations and orientations within an enzyme active site, we have incorporated site-specific thiocyanate vibrational probes into multiple positions within bacterial ketosteroid isomerase. A battery of X-ray crystallographic, vibrational Stark spectroscopy, and NMR studies revealed electrostatic field heterogeneity of 8 MV/cm between active site probe locations and widely differing sensitivities of discrete probes to common electrostatic perturbations from mutation, ligand binding, and pH changes. Electrostatic calculations based on active site ionization states assigned by literature precedent and computational pK(a) prediction were unable to quantitatively account for the observed vibrational band shifts. However, electrostatic models of the D40N mutant gave qualitative agreement with the observed vibrational effects when an unusual ionization of an active site tyrosine with a pK(a) near 7 was included. UV-absorbance and (13)C NMR experiments confirmed the presence of a tyrosinate in the active site, in agreement with electrostatic models. This work provides the most direct measure of the heterogeneous and anisotropic nature of the electrostatic environment within an enzyme active site, and these measurements provide incisive benchmarks for further developing accurate computational models and a foundation for future tests of electrostatics in enzymatic catalysis.
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