Cholinesterase Structure Identification of Residues and Domains Affecting Organophosphate Inhibition and Catalysis

摘要

Studies on the mechanism of inhibition of acetyicholinesterase by nerve agents and its reactivation by oximes were investigated in order to develop improved antidotal therapies. The approach requires an understanding of the structure of the target site of acetyicholinesterase, the mechanisms of its inactivation and reactivation, and the structural determinants of ligand specificity for the target sites. Through the use of enantiomeric organophosphates, selected oximes and site-specific mutants of acetyicholinesterase, we have devised oxime-acetyicholinesterase binary combinations that, when in plasma, should be catalytic scavengers of circulating nerve agents. The efficacy of the particular combinations depends on the structure of the offending nerve agent. Through mechanistic analyses, kinetic studies and the X-ray crystal structures of the organophosphate- enzyme conjugates, we are beginning to understand the structural basis for reactivation. In other studies, through cysteine substitution mutagenesis, we have been able to place fluorophores at selective locations in the enzyme. Analyses of spectroscopic properties of the conjugated fluorophores, along with studies of decay of fluorescence anisotropy, have yielded important information on enzyme microenvironment in the vicinity of the fluorophore, segmental motion of the side chain and the flexibility of the alpha carbon chain of the enzyme. This work suggests that the enzyme crystal structure does not reveal the conformational states of the enzyme, particularly the open gorge conformation, and detailed analysis of conformation of acetylcholinesterase in solution through spectroscopy is warranted.