Interplay Between Substrate Polarity and Protein Dynamics in Evolved Kemp Eliminases

Extensive molecular dynamics (MD) simulations on designed and evolved enzymes for the Kemp Elimination were performed. Over thirty different systems including combinations of three protein scaffolds used in computational design (KE70, KE59 and HG3), several directed evolution variants and ligands (5‐nitrobenzo[d]isoxazole, and 6‐nitro‐1H‐benzo[d][1,2,3]triazole), were screened computationally. The study was focused mostly on computational and evolved variants for which X‐ray structures are available. MD simulations were used to monitor the organization of the active sites over time periods of 100 ns. A general trend was observed in the simulations: the optimal organization of active site residues, corresponding to the most active catalysts, is only maintained with polarized, TS‐like substrates, reinforcing the crucial role of protein dynamics and electrostatic preorganization for efficient biocatalysis. Implications for enzyme design protocols are discussed. Merging enzyme dynamics and electrostatics: Molecular dynamics simulations were performed on different lineages of computationally designed and laboratory‐evolved Kemp eliminases. Various protein scaffolds, all of which were crystallographically characterized, and ligands with different polarities, were explored. Results underscore the importance of electrostatic preorganization in biocatalysis, highlighting trends toward optimal active site organization with specific substrates, with implications for enzyme design.