Kemp elimination catalysts by computational enzyme design

TitleKemp elimination catalysts by computational enzyme design
Publication TypeJournal Article
Year of Publication2008
AuthorsRöthlisberger, D., Khersonsky O., Wollacott A. M., Jiang L., DeChancie J., Betker J., Gallaher J. L., Althoff E. A., Zanghellini A., Dym O., Albeck S., Houk K. N., Tawfik D. S., & Baker D.
Date Published2008 May 8
KeywordsAlgorithms, Amino Acid Motifs, Binding Sites, Catalysis, Computational Biology, Computer Simulation, Crystallography, X-Ray, Directed Molecular Evolution, Drug Design, Drug Evaluation, Preclinical, Enzymes, Kinetics, Models, Chemical, Models, Molecular, Primary Publication, Protein Engineering, Quantum Theory, Sensitivity and Specificity

The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10(5) and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in k(cat)/K(m) (k(cat)/K(m) of 2,600 M(-1)s(-1) and k(cat)/k(uncat) of >10(6)). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.

Alternate JournalNature
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