Notably, we found that the double mutant D253E-H254R exhibits a 19-fold increase in activity over WT. Also, D253E and D253E-I255G enhanced the enzyme activity by 10-and 4.8-fold, respectively. In contrast, I255S did not improve the enzymatic hydrolysis of paraoxon significantly. These results exhibit a similar trend to the computational findings and suggest that hydrogen bonding within the OPH active site is important for the function of the protein and the hydrolysis reaction.