Overall, the binding free energies appear sufficiently converged with reasonable uncertainties (Fig- and). Three designed mutants show more favorable binding free energies to WT OPH with an improvement of 0.63 kcal/mol, 0.26 kcal/mol, and 1.63 kcal/mol for D253E, D253E-H254R, and D253E-I255G, respectively. These findings confirm that the single and the double mutations we introduced here enhance the stability of the substrate in the OPH active site. Some of our other calculations, however, show that the single mutants (H254S and I255S) we designed to alter the property of the side chains at positions 254 and 255 failed to improve the binding of paraoxon to the protein. The calculated binding free energies of the mutants I255S (-20.44 kcal/mol) and H254S (-17.61 kcal/mol) are less favorable than that of the WT complex (-20.70 kcal/mol).