Cholinesterase Structure: Identification of Mechanisms and Residues Involved in Organophosphate Inhibition and Enzyme Reactivation
Final rept. 15 Apr 2002-14 Apr 2005
CALIFORNIA UNIV SAN DIEGO LA JOLLA
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Studies on the structural of acetylcholinesterase AChE as a target of organophosphate toxicity continue and have yielded several leads of significance and practical outcomes. First, studies on oxime reactivation reveal the importance of achieving a suitable angle of attack for the oxime within the confines of the active center gorge. Through the use of mutant AChE-oxime combinations, oxime-assisted catalytic turnover of organophosphates can be achieved such that mutant AChE can be employed with oximes as a catalytic scavenger. Second, through cysteine substitution mutagenesis and acrylodan labeling we have developed a fluorescent enzyme whose emission spectrum changes upon conjugation with organophosphate. These enzymes are now being immobilized and developed as remote sensors for AChE inhibition. Third, we have developed mass spectrometry methods to detect directly the organophosphate conjugates with AChE. Lastly, we have developed several transgenic knock-out animal strains that enable us to study the roles cholinesterase inhibition centrally and in the periphery play in organophosphate toxicity and whether the antidotal actions of oximes arise solely through reactivation.