Investigating the structural influence of surface mutations on acetylcholinesterase inhibition by organophosphorus compounds and oxime reactivation
Introduction
To minimize the toxicity of organophosphate (OP) inhibitors, binary mixtures of mammalian AChE and oximes of varying structure were recently considered as catalytic bioscavengers promoting the catalysis of the OP in plasma before it reacts with the target site in skeletal muscle or the nervous system [1]. Our goal is to generate site-specific mutations in AChE protein sequence in order to convert human AChE into catalytic scavenger that, when coupled with an oxime, will inactivate multiple OP molecules per one molecule of scavenger.
Human AChE wild type and D132H, D134H_E202Q, and D134H_F338A mutants were characterized and investigated for inhibition by OPs (an insecticide and nerve agent analogues; Fig. 1) and reactivation of the phosphylated enzymes with 2PAM and HI6. The rationale for selecting these substitution positions was based on D134H being a naturally occurring SNP in humans and that E202Q and F338A mutations slow aging of OP inhibited AChEs [2], [3].
Section snippets
Materials and methods
Nerve agent analogues of cyclosarin (methylphosphonic acid 3-cyano-4-methyl-2-oxo-2H-coumarin-7-yl ester cyclohexyl ester), VX (methylphosphonic acid 3-cyano-4-methyl-2-oxo-2H-coumarin-7-yl ester ethyl ester), soman (methylphosphonic acid 3-cyano-4-methyl-2-oxo-2H-coumarin-7-yl ester pinacolyl ester) were synthesized as previously described [4], as well as SP-Cycloheptyl methyl phosphonyl thiocholine (SPCHMP) and RP-Isopropyl methyl phosphonyl thiocholine (RPIPMP) [5]. O,O-Diethyl
Results and discussion
Inhibition of human D134H with paraoxon and an analogue of cyclosarin was 2–8 times slower than that of human wt (Table 1), whereas inhibition by soman and analogues of VX was not affected by D134H substitution. Introduction of additional substitutions in two double mutants further slowed inhibition up to two orders of magnitude.
2PAM reactivation of paraoxon inhibited human D134H is, on the other hand, 6 times faster than human wt (Table 2). Our previous study indicated that differential
Conflict of interest statement
None declared.
Acknowledgements
This study was supported by fellowships from IIE and The Fulbright Program, and the Scientific and Technical Research Council of Turkey to Tuba Kucukkilinc and by NIH NINDS U01NS58046 to Palmer Taylor.
References (8)
- et al.
Acetylcholinesterase: converting a vulnerable target to a template for antidotes and detection of inhibitor exposure
Toxicology
(2007) - et al.
The role of glutamate-199 in the aging of cholinesterase
Biochem. Biophys. Res. Commun.
(1993) - et al.
Asymmetric fluorogenic organophosphates for the development of active organophosphate hydrolases with reversed stereoselectivity
Toxicology
(2007) - et al.
Chiral reactions of acetylcholinesterase probed with enantiomeric methylphosphonothioates. Noncovalent determinants of enzyme chirality
J. Biol. Chem.
(1989)
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