Molecular Gating of an Engineered Enzyme Captured in Real Time
| Autor: | Koen Beerens, Michaela Slanska, Mariana Amaro, David Bednar, Zbynek Prokop, Martin Hof, Šárka Bidmanová, Jan Brezovsky, Jan Sykora, Avisek Ghose, Jiri Damborsky, Piia Kokkonen |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Hydrolases Protein Conformation Kinetics Fluorescence correlation spectroscopy Molecular Dynamics Simulation Protein Engineering Biochemistry Catalysis Fluorescence spectroscopy 03 medical and health sciences Molecular dynamics Colloid and Surface Chemistry Catalytic Domain biology Chemistry Protein dynamics Active site General Chemistry Combinatorial chemistry Sphingomonadaceae 030104 developmental biology Catalytic cycle Mutation Biocatalysis biology.protein Haloalkane dehalogenase |
| Zdroj: | Journal of the American Chemical Society. 140:17999-18008 |
| ISSN: | 1520-5126 0002-7863 |
| DOI: | 10.1021/jacs.8b09848 |
| Popis: | Enzyme engineering tends to focus on the design of active sites for the chemical steps, while the physical steps of the catalytic cycle are often overlooked. Tight binding of a substrate in an active site is beneficial for the chemical steps, whereas good accessibility benefits substrate binding and product release. Many enzymes control the accessibility of their active sites by molecular gates. Here we analyzed the dynamics of a molecular gate artificially introduced into an access tunnel of the most efficient haloalkane dehalogenase using pre-steady-state kinetics, single-molecule fluorescence spectroscopy, and molecular dynamics. Photoinduced electron-transfer fluorescence correlation spectroscopy (PET-FCS) has enabled real-time observation of molecular gating at the single-molecule level with rate constants (k(on) = 1822 s(-1), k(off) = 60 s(-1)) corresponding well with those from the pre-steady-state kinetics (k(-1) = 1100 s(-1), k(1) = 20 s(-1)). The PET-FCS technique is used here to study the conformational dynamics in a soluble enzyme, thus demonstrating an additional application for this method. Engineering dynamical molecular gates represents a widely applicable strategy for designing efficient biocatalysts. |
| Databáze: | OpenAIRE |
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