title: Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters.
abstract: Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the (122)GFYS(125) peptide segment, resulting in (122)HFQT(125). This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k(cat)/K(m) ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.
authors.affiliation: Département de Toxicologie, Centre de Recherches du Service de Santé des Armées, BP 87, 38702 La Tronche Cedex, France. tpoyot@crssa.net
authors.forename: Thomas
authors.initials: T
authors.lastname: Poyot
authors.forename: Florian
authors.initials: F
authors.lastname: Nachon
authors.forename: Marie-Thérèse
authors.initials: MT
authors.lastname: Froment
authors.forename: Mélanie
authors.initials: M
authors.lastname: Loiodice
authors.forename: Stacy
authors.initials: S
authors.lastname: Wieseler
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Schopfer
authors.forename: Oksana
authors.initials: O
authors.lastname: Lockridge
authors.forename: Patrick
authors.initials: P
authors.lastname: Masson
mesh_terms: D000110:Acetylcholinesterase / Q000235:genetics* / Q000378:metabolism*; D000122:Acetylthiocholine / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D017838:Bungarus; D004390:Chlorpyrifos / Q000031:analogs & derivatives / Q000494:pharmacology; D004222:Disulfoton / Q000494:pharmacology; D004456:Echothiophate Iodide / Q000378:metabolism / Q000494:pharmacology; D007531:Isoflurophate / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010261:Paraoxon / Q000378:metabolism / Q000494:pharmacology
chemical_list: D009943:Organophosphorus Compounds; D007531:Isoflurophate; D004222:Disulfoton; D000122:Acetylthiocholine; C009618:O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphate; D004456:Echothiophate Iodide; D000110:Acetylcholinesterase; D004390:Chlorpyrifos; D010261:Paraoxon