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Related Experiment Video

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Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

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Kemp elimination catalysts by computational enzyme design.

Daniela Röthlisberger¹, Olga Khersonsky, Andrew M Wollacott

¹Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.

|March 21, 2008

Summary

Researchers computationally designed novel enzymes to catalyze reactions previously uncatalyzed by natural biocatalysts. This protein engineering breakthrough achieved significant rate enhancements, demonstrating a powerful new approach for creating custom enzymes.

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Area of Science:

Biochemistry
Protein Engineering
Computational Biology

Background:

Designing novel enzymes for non-natural reactions is a significant challenge in protein engineering.
Understanding enzyme catalysis is crucial for advancing biocatalysis.
The Kemp elimination serves as a model reaction for studying proton transfer from carbon.

Purpose of the Study:

To computationally design and create novel enzymes capable of catalyzing the Kemp elimination reaction.
To validate the catalytic activity and structural accuracy of the designed enzymes.
To enhance the performance of computationally designed enzymes using directed evolution.

Main Methods:

Computational protein design utilizing two distinct catalytic motifs.
In vitro experimental validation of enzyme activity, including rate enhancement measurements.
Mutational analysis to confirm the role of designed active sites.
High-resolution crystal structure determination.
Directed evolution (in vitro evolution) to improve enzyme efficiency.

Main Results:

Successfully designed eight novel enzymes catalyzing the Kemp elimination with rate enhancements up to 10^5 and multiple turnovers.
Mutational analysis and crystal structure confirmed the accuracy and functionality of the computationally designed active sites.
Directed evolution increased the catalytic efficiency (kcat/Km) by over 200-fold, reaching 2,600 M^-1s^-1, with kcat/kuncat > 10^6.

Conclusions:

Computational protein design combined with directed evolution is a powerful strategy for creating new enzymes.
The designed enzymes demonstrate high catalytic efficiency and structural accuracy.
This approach holds significant promise for the future development of novel biocatalysts for various applications.