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Updated: Jun 29, 2025
10:45
Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip
Published on: March 20, 2021
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Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography
Nathan Smith1, Medhanjali Dasgupta1, David C Wych2,3
1Department of Biochemistry and Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
Science Advances
|March 27, 2024
Summary
Enzymes utilize dynamic structures for catalysis. This study used advanced X-ray crystallography to reveal how isocyanide hydratase (ICH) enzyme structure changes during catalysis, driven by residue ionization and protein motion.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Enzymes exist as dynamic structural ensembles, crucial for catalysis but challenging to study.
- Understanding these ensembles is key to characterizing enzyme mechanisms.
Purpose of the Study:
- To investigate the dynamic structural changes of isocyanide hydratase (ICH) during catalysis.
- To elucidate the role of residue ionization and protein motion in enzyme function.
Main Methods:
- Time-resolved mix-and-inject serial crystallography using an X-ray free electron laser.
- Determination of enzyme structures at multiple pH values.
- In crystallo molecular dynamics simulations and time-resolved electron density mapping.
Main Results:
- Observed catalysis in a designed ICH mutant, enhancing sampling of minor conformations.
- Formation of the thioimidate intermediate selected for catalytically competent substates.
- Aspartic acid 17 ionization was shown to trigger conformational changes and water entry for hydrolysis.
Conclusions:
- Isocyanide hydratase couples residue ionization to catalysis-activated protein motions.
- Demonstrated a mechanism of electrostatic control over enzyme dynamics.
- Provided insights into the dynamic nature of enzyme active sites during catalysis.
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