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Distal mutations enhance catalysis in designed enzymes by facilitating substrate binding and product release
Niayesh Zarifi1,2, Pooja Asthana3, Hiva Doustmohammadi4
1Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario, Canada.
Nature Communications
|September 30, 2025
Summary
Distal amino-acid mutations enhance enzyme catalysis by improving substrate binding and product release. These findings reveal that optimal enzyme function requires both an organized active site and dynamic structural regulation by distant residues, crucial for enzyme design.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Structural biology
Background:
- The function of amino-acid residues distant from an enzyme's active site is not well understood.
- Understanding these distal residues is key to elucidating the complete enzyme catalytic cycle.
Purpose of the Study:
- To investigate how mutations in distal amino-acid residues influence enzyme catalysis.
- To compare the effects of active-site versus distal mutations on enzyme activity.
Main Methods:
- Engineering de novo Kemp eliminase mutants with active-site or distal mutations.
- Utilizing kinetic analyses, X-ray crystallography, and molecular dynamics simulations.
- Analyzing the impact of mutations on substrate binding, chemical transformation, and product release.
Main Results:
- Active-site mutations enhance chemical transformation by creating preorganized active sites.
- Distal mutations facilitate substrate binding and product release by tuning protein dynamics.
- Distal mutations widen the active-site entrance and reorganize surface loops, improving overall enzyme activity.
Conclusions:
- A well-organized active site is necessary but not sufficient for optimal enzyme catalysis.
- Distal residues play critical roles in enhancing catalytic efficiency by modulating enzyme dynamics.
- These findings provide valuable insights for the rational design of enzymes with improved catalytic properties.
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