Time-resolved crystallography reveals allosteric communication aligned with molecular breathing

Area of Science:

• Biochemistry and Structural Biology
• Enzymology
• Protein Dynamics

Background:

• Understanding enzyme function requires linking protein structure to dynamic changes.
• Fluoroacetate dehalogenase is an enzyme crucial for metabolic processes.
• Visualizing enzyme catalysis at high temporal resolution remains challenging.

Purpose of the Study:

• To visualize the dynamic structural changes of fluoroacetate dehalogenase during its catalytic cycle.
• To correlate molecular breathing motions with enzymatic activity.
• To understand the role of protein dynamics and entropy in catalysis.

Main Methods:

• Employed time-resolved serial synchrotron crystallography to capture rapid structural changes.
• Collected data at 18 time points spanning 30 milliseconds to 30 seconds.
• Analyzed four turnover cycles of the irreversible enzymatic reaction.

Main Results:

• Observed sequential steps: substrate binding, covalent intermediate formation, water molecule activation, and product release.
• Identified subtle protein structural rearrangements and dynamic water molecule positioning throughout catalysis.
• Visualized half-of-the-sites reactivity and correlated molecular breathing motions with catalytic steps.

Conclusions:

• Enzyme catalysis involves dynamic and entropic contributions from repetitive protein framework changes.
• Molecular breathing motions, triggered by enzyme-ligand interactions, are integral to the catalytic machinery.
• Time-resolved crystallography provides unprecedented insight into enzyme reaction mechanisms.