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Butyrylcholinesterase (BChE) is considered as an efficient stoichiometric antidote against organophosphorus (OP) poisons. Moreover, recently several BChE variants capable of full cycle of catalytic destruction of OP pesticide paraoxon were obtained using ultrahigh-throughput combinatorial screening. These variants harbour 5 substitutions in the acyl-binding loop, however, cause for the emerged reactivation ability is ambiguous. In this study we determined the detailed molecular mechanism of organophosphatase activity mediated by these BChE variants using both classical and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. Detailed analysis of QM/MM simulations revealed that histidine residues introduced into the acyl-binding loop are always located in close proximity with the aspartate 70 which is located at the entrance of the binding site. Histidine residue acts as general base thus leading to the activation of the attacking water molecule and subsequent SN2 inline hydrolysis which finally results in BChE reactivation. The combination of this aspartate, introduced histidine and water molecule resembles canonical catalytic triad found in active centers of various proteases as well as original active site of BChE itself. Aspartate’s carboxyl group activates histidine residue by altering pKa of the latter, which in turn promotes the activation of water molecule in terms of its nucleophilicity. Observed re-protonation of catalytic serine 198 from catalytic histidine 438 recovers initial configuration of the enzyme’s active center, facilitating next catalytic cycle. Estimated energy barriers are in good agreement with experimental data thus supporting our mechanistic findings. This data suggest that BChE with multiple substitutions in principle may seize organophosphatase activity and gives new ideas on design strategies to finally produce an efficient organophosphate-hydrolyzing enzyme. Deciphering of molecular mechanisms by presented top-down approach may significantly improve our knowledge of enzyme function, propose new strategies for enzyme design and open new horizons in generation of catalytic bioscavengers against OP poisons.