Fig. 6. (A) A comparison of calculated (ΔG‡calc) and experimental (ΔG‡exp) activation free energies for the Kemp elimination of 5-nitrobenzisoxazole by the GNCA4-WT β-lactamase, and a series of its active site mutants (see also Table 1). (B) The electrostatic contributions of individual residues to the calculated activation free energies (ΔΔG‡elec) for the Kemp elimination of 5-nitrobenzisoxazole by the GNCA4-WT β-lactamase (treated as the baseline ‘wild-type’ enzyme in this work). All values were obtained by applying the linear response approximation (LRA)83,84 to the calculated EVB trajectories, as in our previous works,85–87 and scaled assuming a dielectric constant of 4 for the highly hydrophobic environment of the de novo active site of this β-lactamase (Fig. 1). For the correlation between calculated and experimental values, see Fig. S6.† .