The relative protein–ligand binding free energy, ΔΔG°, corresponds to the difference in binding affinities between the initial and the target (WT) enzyme, i.e., , which is equal to the difference in alchemical free energies between the bound and unbound states, i.e., ΔG^bound_BAR − ΔG^unbound_BAR (see Table S4). The ΔΔG° values reflect the concomitant amino-acid protonation state change in both monomers of the homodimeric enzyme. The values supplied in parentheses represent the relative protein–ligand binding free energy per monomer. All energies are in kcal mol⁻¹.

Transformation	Substrate	ΔΔG°
HD41 → HE41	Ketoamide	−2.7 ± 0.3 (−1.3 ± 0.3)
HE163 → HP163	Ketoamide	+6.7 ± 1.4 (+3.4 ± 1.4)
HD41 → HE41	N3	+1.5 ± 0.1 (+0.8 ± 0.1)
HE163 → HP163	N3	+3.9 ± 1.0 (+1.9 ± 1.0)