Precision is essential for efficient catalysis in an evolved Kemp eliminase

A computationally designed enzyme that was evolved to accelerate a chemical reaction 6 × 10 8 -fold approaches the exceptional efficiency of highly optimized natural enzymes and provides valuable lessons for the creation of more sophisticated artificial catalysts. Fine-tuning enzyme catalysis Enzymes selectively stabilize the rate-limiting transition state of the catalysed reaction relative to the ground state. Previous attempts to exploit this idea, for example by using transition-state analogues to elicit antibodies with catalytic activities, have generally failed to deliver true enzyme-like catalysts. In this study, the authors engineered and evolved a computationally designed protein catalyst for the Kemp elimination, a well-studied model system for proton transfer from carbon, into an artificial enzyme that accelerates the chemical reaction 6 × 10 8 fold, approaching the exceptional efficiency of highly optimized natural enzymes. A high-resolution X-ray crystal structure of the evolved enzyme indicates that well-known catalytic strategies, such as shape complementarity and precisely placed catalytic groups, were successfully harnessed to afford such high rate accelerations. Linus Pauling established the conceptual framework for understanding and mimicking enzymes more than six decades ago 1 . The notion that enzymes selectively stabilize the rate-limiting transition state of the catalysed reaction relative to the bound ground state reduces the problem of design to one of molecular recognition. Nevertheless, past attempts to capitalize on this idea, for example by using transition state analogues to elicit antibodies with catalytic activities 2 , have generally failed to deliver true enzymatic rates. The advent of computational design approaches, combined with directed evolution, has provided an opportunity to revisit this problem. Starting from a computationally designed catalyst for the Kemp elimination 3 —a well-studied model system for proton transfer from carbon—we show that an artificial enzyme can be evolved that accelerates an elementary chemical reaction 6 × 10 8 -fold, approaching the exceptional efficiency of highly optimized natural enzymes such as triosephosphate isomerase. A 1.09 Å resolution crystal structure of the evolved enzyme indicates that familiar catalytic strategies such as shape complementarity and precisely placed catalytic groups can be successfully harnessed to afford such high rate accelerations, making us optimistic about