In this study, each simulation was conducted in five different replicas. Each replica underwent an FEP simulation consisting of 31 frames, where the coupling parameter 𝜆 was incrementally changed in 31 equal steps of 0.03. Each MD frame involved a 20 ps equilibration under the potential 𝑉(𝜆) . The five replicas began from distinct initial configurations obtained through five successive 20 ps equilibration steps. For the reference reaction in water, the serine and histidine residues were Cα-methylated. During the MD simulations, a force of 10 kcal/mol/Å² was applied to keep the Cα atoms in their original positions. Otherwise, the FEP protocol in water mirrored that in the enzyme.