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Dissecting the Conformational Dynamics-Modulated Enzyme Catalysis with Single-Molecule FRET
Shaowen Wu1, Jianwei Liu1, Wenning Wang1
1Key Laboratory of Medical Epigenetics and Metabolism, Department of Chemistry and Institutes of Biomedical Sciences , Fudan University , Shanghai 200433 , P.R. China.
The Journal of Physical Chemistry. B
|May 17, 2018
Summary
This study introduces a new method using single-molecule Förster resonance energy transfer (smFRET) to simultaneously analyze enzyme conformational changes and catalytic reactions, revealing previously unmeasurable kinetic parameters for enzyme mechanisms.
Area of Science:
- Biochemistry
- Biophysics
- Enzymology
Background:
- Enzyme activity is modulated by conformational changes during catalysis.
- Single-molecule technology offers insights into enzyme mechanisms.
- Simultaneous monitoring of reaction cycles and conformational changes is challenging.
Purpose of the Study:
- To develop a method for extracting all kinetic parameters from enzyme studies.
- To enable simultaneous analysis of chemical reactions and conformational dynamics.
- To quantitatively reveal enzyme catalytic mechanisms coupled with conformational changes.
Main Methods:
- Utilizing comprehensive single-molecule Förster resonance energy transfer (smFRET) measurements.
- Applying kinetic model analysis to smFRET data.
- Solving rate equations with smFRET-derived conformational populations and transition rates.
Main Results:
- Successfully extracted previously undetectable kinetic parameters.
- Demonstrated the method's efficacy with a MalK2 ATPase case study.
- Revealed quantitative mechanisms of enzyme catalytic reactions and coupled conformational dynamics.
Conclusions:
- The developed method allows for comprehensive kinetic parameter extraction in enzyme catalysis.
- This strategy provides a quantitative understanding of enzyme conformational dynamics.
- The approach is broadly applicable to studying other enzymes with conformational fluctuation-coupled catalysis.
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