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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 theMycobacterium tuberculosisβ-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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Reaction Initiation in Enzyme Crystals by Diffusion of Substrate
Ever since the first structure of an enzyme, lysozyme, was solved, scientists have been eager to explore how these molecules perform their catalytic function. There has been an overwhelmingly large body of publications that report the X-ray structures of enzymes determined after substrate and ligand binding. None of them truly show the structures of an enzyme working freely through a sequence of events that range from the formation of the enzyme–substrate complex to the dissociation of the product. The technical difficulties were too severe. By 1969, Sluyterman and de Graaf had pointed out that there might be a way to start a reaction in an enzyme crystal by diffusion and following its catalytic cycle in its entirety with crystallographic methods. The crystal only has to be thin enough so that the diffusion is not rate limiting. Of course, the key questions are as follows: How thin should the crystal be? Will the existing X-ray sources be able to collect data from a thin enough crystal fast enough? This review shines light on these questions.
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- Award ID(s):
- 1231306
- PAR ID:
- 10588338
- Publisher / Repository:
- Crystals
- Date Published:
- Journal Name:
- Crystals
- Volume:
- 10
- Issue:
- 2
- ISSN:
- 2073-4352
- Page Range / eLocation ID:
- 116
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/cryst10020116
