Evolution promotes transition-state-like conformations with enhanced basicity and electric fields in the substrate complex of a designer enzyme

Enzymes can be computationally designed for an increasing range of reactions, yet the catalytic efficiencies achieved are typically well below those of natural biocatalysts. Directed evolution can narrow this gap, often improving activity through mutations with non-obvious effects on catalysis. Here, we dissect how directed evolution improved a de novo designed Kemp eliminase by comparing the ground-state and transition-state ensembles of the designed and evolved variants. Extensive molecular dynamics simulations and QM/MM reaction barrier calculations identify subtle but important differences that modulate reactivity. Simulations show that reaction proceeds via a specific transition-state-like conformation, characterized by desolvation of the catalytic base and an organized active-site electric field. Barriers correlate strongly with the reaction energy of proton transfer, indicating that desolvating the catalytic base in the ligand-bound complex raises its proton affinity and thereby lowers the barrier. Evolution enriches the ground-state ensemble in reactive conformations, minimizing the reorganization required to reach the transition state. After evolution, formation of the transition-state-like conformation causes less disruption to the protein, which maintains coordinated motions. This is consistent with the emergence of a negative activation heat capacity with evolution. Efficient catalysis requires a ground-state ensemble that resembles the transition state, an objective that enzyme design should pursue alongside geometric and electrostatic complementarity for the transition state.