Journal Article

Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography

Koliyadu, Jayanath, Fromme, Petra, Tolstikova, Alexandra, Schwander, Peter, Gelisio, Luca, Vakili, Mohammad, Doerner, Katarina, Kloos, Marco, Mariani, Valerio, Xu, Weijun, Pandey, Suraj, Schmidt, Marius, Spence, John C. H., Kirkwood, Henry, Yang, Jay-How, Sadri, Alireza, Martin-Garcia, Jose M., Bean, Richard, Pollack, Lois, Malla, Tek Narsingh, Olmos, Jose L., Calvey, George, Chapman, Henry N., Koua, Faisal H. M., Yefanov, Oleksandr, Sato, Tokushi, Mancuso, Adrian P., Katz, Andrea M., Zielinski, Kara A., Ourmazd, Abbas, Frank, Matthias, Awel, Salah, Barty, Anton, Bajt, Sasa, Nelson, Garrett, Phillips, George N., Poudyal, Ishwor, Mills, Grant, Jernigan, Rebecca, Miller, Mitchell D.

2021

Overview

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.

• Publisher: International Union of Crystallography
• Subject: Chemistry / Crystallography / Materials Science