For H254S, our kinetic measurements show an improvement in activity by 5.2 fold compared to the WT form. This enhancement was not captured in our binding free energy predictions and might be due to the limitations of the computational method. One major limitation is the use of implicit solvent since the water structure could affect changes in protein-ligand complexes. On the other hand, our MD simulations performed in explicit solvent align with the experimental results, supporting our speculations regarding the solvent. MD simulations show that H254S induces a hydrogen bond that stabilizes one of the Zn(II) coordinators, which could explain the enhanced catalytic activity of the enzyme. An additional limitation, of course, is that we are experimentally measuring activity, which is a function of more than just the binding free energy.