Oxime-assisted reactivation of tabun-inhibited acetylcholinesterase analysed by active site mutations.

The antidotal property of oximes is attributed to their ability to reactivate acetylcholinesterase (AChE) inhibited by organophosphorus compounds (OP) such as pesticides and nerve warfare agents. Understanding their interactions within the active site of phosphylated AChE is of great significance for the search for more efficient reactivators, especially in the case of the most resistant OP to reactivation, tabun. Therefore, herein we studied the interactions and reactivation of tabun-inhibited AChE by site-directed mutagenesis and a series of bispyridinium oximes. Our results indicated that the replacement of aromatic residues with aliphatic ones at the acyl pocket and choline binding site mostly interfered with the stabilisation of the oxime's pyridinium ring(s) within the active site gorge needed to obtain the proper orientation of the oxime group toward the phosphorylated active site serine. However, in the case of W286A, the mutation in the peripheral binding site by preventing a π-π interaction with one of the oxime's pyridinium rings allowed a more favourable position of the oxime for a nucleophilic attack on the phosphorylated catalytic serine. The mutation resulted in a 2-5 fold increase in the reactivation rates when compared to the AChE wild type. Therefore, it seems that aromatic amino acids at the peripheral binding site presented a limitation in bispyridinium oxime reactivation efficiency of tabun-phosphorylated AChE. Moreover, this is further corroborated by the reactivation by mono-pyridinium oxime 2-PAM, in which mutations at the peripheral site did not influence either the affinity or reactivation of tabun-inhibited AChE.