Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography.
IUCrJ
; 8(Pt 6): 878-895, 2021 Nov 01.
Article
en En
| MEDLINE
| ID: mdl-34804542
ABSTRACT
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-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis ß-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 66â
ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.
European X-ray Free-Electron Laser; X-ray crystallography; antibiotic resistance; ceftriaxone; drug discovery; enzyme kinetics; enzyme mechanisms; irreversible inhibition; megahertz pulse-repetition rate; mix-and-inject serial crystallography; protein structure determination; serial femtosecond crystallography; substrate diffusion in crystals; sulbactam; ß-lactamases
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1
Colección:
01-internacional
Base de datos:
MEDLINE
Idioma:
En
Revista:
IUCrJ
Año:
2021
Tipo del documento:
Article
País de afiliación:
Estados Unidos