期刊
IUCRJ
卷 8, 期 -, 页码 878-895出版社
INT UNION CRYSTALLOGRAPHY
DOI: 10.1107/S2052252521008125
关键词
substrate diffusion in crystals; antibiotic resistance; beta-lactamases; enzyme kinetics; irreversible inhibition; mix-and-inject serial crystallography; serial femtosecond crystallography; European X-ray Free-Electron Laser; megahertz pulse-repetition rate; protein structure determination; drug discovery; ceftriaxone; sulbactam; X-ray crystallography; enzyme mechanisms
资金
- National Science Foundation Science and Technology Center 'BioXFEL' [STC-1231306]
- US Department of Energy, Office of Science, Basic Energy Sciences [DE-SC0002164]
- National Science Foundation [1551489, DBI-2029533, 1450681, 1565180]
- National Institutes of Health [R01 GM117342-0404, R01 GM095583]
- Biodesign Center for Applied Structural Discovery at ASU
- US Department of Energy through Lawrence Livermore National Laboratory [DE-AC52-07NA27344]
- Cornell Molecular Biophysics Training Program [NIH T32-GM008267]
- Cluster of Excellence 'CUI: Advanced Imaging of Matter' of the Deutsche Forschungsgemeinschaft (DFG) [EXC 2056, 390715994]
- Gottfried Wilhelm Leibniz Program of the DFG
- 'X-probe' project - European Union 2020 Research and Innovation Program under Marie Sklodowska-Curie grant [637295]
- European Research Council, 'Frontiers in Attosecond X-ray Science: Imaging and Spectroscopy (AXSIS)' [ERC-2013-SyG 609920]
- Human Frontiers Science Program [RGP0010 2017]
- AXSIS project - European Research Council under the European Union Seventh Framework Program (FP/2007-2013)/ERC Grant [609920]
- Division of Computing and Communication Foundations
- Direct For Computer & Info Scie & Enginr [1551489] Funding Source: National Science Foundation
- Div Of Biological Infrastructure
- Direct For Biological Sciences [1565180] Funding Source: National Science Foundation
This study utilized the high repetition rate of EuXFEL and mix-and-inject technology to observe the initial phase of ceftriaxone binding to Mycobacterium tuberculosis beta-lactamase in real time, demonstrating the importance of this technique for biomedically relevant research.
Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-andinject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis beta-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme-ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.
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