Probing the role of amino acids in oxime-mediated reactivation of nerve agent-inhibited human acetylcholinesterase

•Wild-type and mutant human AChEs were inhibited with GA, GB, GF, VX, or VR.•Reactivation rate constants for 2-PAM, MMB4, HI-6, and HLö-7 were compared.•Y72, Y124 and W286 were important only in reactivation by bis-pyridinium oximes.•E202 was important for reactivation by all oximes.•Human and bovin...

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Published in:Toxicology in vitro 2015-03, Vol.29 (2), p.408-414
Main Authors: Chambers, Carolyn, Luo, Chunyuan, Tong, Min, Yang, Yerie, Saxena, Ashima
Format: Article
Language:English
Subjects:
Acetylcholinesterase - genetics
Acetylcholinesterase - metabolism
Amino Acids - genetics
Animals
Cattle
Chemical Warfare Agents - toxicity
CHO Cells
Cholinesterase Reactivators - pharmacology
Cricetulus
Human AChE mutants
Humans
In vitro study
Nerve agents
Organophosphorus Compounds - toxicity
Oximes
Oximes - pharmacology
Pralidoxime Compounds - pharmacology
Pyridinium Compounds - pharmacology
Reactivation
Site-directed mutagenesis
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Summary:•Wild-type and mutant human AChEs were inhibited with GA, GB, GF, VX, or VR.•Reactivation rate constants for 2-PAM, MMB4, HI-6, and HLö-7 were compared.•Y72, Y124 and W286 were important only in reactivation by bis-pyridinium oximes.•E202 was important for reactivation by all oximes.•Human and bovine AChEs display similarity with regard to oxime reactivation. In this study, we employed site-directed mutagenesis to understand the role of amino acids in the gorge in oxime-induced reactivation of nerve agent-inhibited human (Hu) acetylcholinesterase (AChE). The organophosphorus (OP) nerve agents studied included GA (tabun), GB (sarin), GF (cyclosarin), VX, and VR. The kinetics of reactivation were examined using both the mono-pyridinium oxime 2-PAM and bis-pyridinium oximes MMB4, HI-6, and HLö-7. The second-order reactivation rate constants were used to compare reactivation of nerve agent-inhibited wild-type (WT) and mutant enzymes. Residues including Y72, Y124 and W286 were found to play important roles in reactivation by bis-pyridinium, but not by mono-pyridinium oximes. Residue Y124 also was found to play a key role in reactivation by HI-6 and HLö-7, while E202 was important for reactivation by all oximes. Residue substitutions of F295 by Leu and Y337 by Ala showed enhanced reactivation by bis-pyridinium oximes MMB4, HI-6, and HLö-7, possibly by providing more accessibility of the OP moiety associated at the active-site serine to the oxime. These results are similar to those observed previously with bovine AChE and demonstrate that there is significant similarity between human and bovine AChEs with regard to oxime reactivation.
ISSN:0887-2333
1879-3177
DOI:10.1016/j.tiv.2014.11.001