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Use of V‐Agent Analogs to Probe the Active Site of Atypical Butyrylcholinesterase from Oryzias latipes
Author(s) -
Kirkpatrick Melanie,
Sweeney Richard,
Kasten Shane,
diTargiani Robert,
Cerasoli Douglas,
Otto Tamara
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.573.6
Subject(s) - butyrylcholinesterase , acetylcholinesterase , nerve agent , chemistry , active site , cholinesterase , oryzias , pharmacology , carboxylesterase , enzyme , stereochemistry , biochemistry , aché , biology , gene
The atypical butyrylcholinesterase (aBuChE) from Oryzias latipes shares approximately 65% sequence similarity to both acetylcholinesterase and butyrylcholinesterase and was studied for its capacity to spontaneously reactivate following inhibition by organophosphorus nerve agents. The full‐length aBuChE protein was expressed and purified from cabbage loopers ( Trichoplusia ni larvae) infected with an orally active form of baculovirus. Like other cholinesterases, aBuChE was inhibited by all G‐ and V‐type nerve agents. Interestingly, aBuChE was able to undergo spontaneous reactivation after inhibition with VR. Mass spectrometry of aBuChE after inhibition with VR confirmed the presence of a covalently bound adduct of the size expected for non‐aged VR on the peptide containing the active site serine. To understand the effect of substrate volume on rates of reactivation, the capacity of aBuChE to bind and spontaneously reactivate after inhibition with five analogs of VX and VR and the nerve agent VS was examined. The data suggest that the ability of aBuChE to spontaneously reactivate after inhibition by V‐agent analogs and the rates of reactivation are related to the structure of the retained group. These results may provide structural information to inform the design of improved oximes to reactivate nerve agent‐inhibited acetylcholinesterase or butyrylcholinesterase, and further suggest that re‐engineering the active site of a cholinesterase could result in enzymes with clinically relevant rates of nerve agent hydrolysis. This research was supported by the Defense Threat Reduction Agency – Joint Science Technology Office, Medical S&T Division.