1. Department of Biochemistry,
2. Biomolecular Structure and Design, and,
3. Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA
4. Department of Biological Chemistry, and,
5. Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot 76100, Israel
6. Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA
7. These authors contributed equally to this work.

Correspondence to: Dan S. Tawfik⁴David Baker^1,2,3 Correspondence and requests for materials should be addressed to D.B. (Email: dabaker@u.washington.edu) or D.S.T. (Email: dan.tawfik@weizmann.ac.il).

Abstract

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 10⁵ 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^-1s^-1 and k _cat/k _uncat of >10⁶). 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.

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