Researchers have designed eight enzymes using computational methods to catalyze the Kemp elimination, a reaction for proton transfer from carbon. These enzymes show significant rate enhancements, up to 10^5, and multiple turnovers. The design involved two catalytic motifs: a carboxylate and a His–Asp dyad. Computational modeling and in vitro evolution were used to optimize the enzymes, resulting in a 200-fold increase in k_cat/K_m. The enzymes were characterized experimentally, showing high catalytic efficiency and multiple turnovers. Structural analysis confirmed the accuracy of the designs. The study highlights the potential of combining computational design with directed evolution to create new enzymes, with applications in biotechnology, biomedicine, and industrial processes. The results demonstrate the effectiveness of computational enzyme design in creating efficient catalysts for reactions not naturally catalyzed by enzymes. The study also shows that the computational design methodology can be generalized to design catalysts for more complex reactions. The combination of computational design and molecular evolution is a powerful approach for creating new enzyme catalysts for a wide range of chemical reactions. The research provides insights into the mechanisms of enzyme catalysis and the design of new biocatalysts.Researchers have designed eight enzymes using computational methods to catalyze the Kemp elimination, a reaction for proton transfer from carbon. These enzymes show significant rate enhancements, up to 10^5, and multiple turnovers. The design involved two catalytic motifs: a carboxylate and a His–Asp dyad. Computational modeling and in vitro evolution were used to optimize the enzymes, resulting in a 200-fold increase in k_cat/K_m. The enzymes were characterized experimentally, showing high catalytic efficiency and multiple turnovers. Structural analysis confirmed the accuracy of the designs. The study highlights the potential of combining computational design with directed evolution to create new enzymes, with applications in biotechnology, biomedicine, and industrial processes. The results demonstrate the effectiveness of computational enzyme design in creating efficient catalysts for reactions not naturally catalyzed by enzymes. The study also shows that the computational design methodology can be generalized to design catalysts for more complex reactions. The combination of computational design and molecular evolution is a powerful approach for creating new enzyme catalysts for a wide range of chemical reactions. The research provides insights into the mechanisms of enzyme catalysis and the design of new biocatalysts.