Our final model system focuses on two engineered Kemp eliminase variants (Figure ) from the KE07 series, with kcat and kcat/Km differences of about 35-fold between the two variants studied here (Figure ), and ~100-fold between the initial design and the final optimized variant from the series. A prior study that combined X-ray crystallography, MD simulations and fluorescence spectroscopy to study these variants identified conformational selection as playing a key role in enhancing enzymatic activity, with the emergence of multiple active site conformations along the directed evolution trajectory, during which the lowest energy active site conformation changed from the "A" to the "C" conformation as the enzyme activity increased (Figure ). Four key residues make up part of the enzyme active site (Figure ), with W50 used to bind the substrate and stabilize the transition state; E101 to act as base (for C-H bond cleavage); Y128 to bind the substrate; and K222 as a hydrogenbond donor to stabilize the phenoxide intermediate. The transition from the "A" to "C" conformational state relates to a flip of the W50 ring to hydrogen bond with E101 and a breaking of the E101-K222 hydrogen bond (Figure ). (A) Corresponding crystal structures of the selected KE07 variants, with mutations sites indicated by spheres and labelled. (B) The kcat and kcat/KM of the two KE07 variants that will be evaluated in this study. (C) The two major active site conformations of the selected KE07 variants, with the key catalytic residues that remain unchanged throughout the DE process labeled.