Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters

Thomas Poyot, Florian Nachon, Marie Thérèse Froment, Mélanie Loiodice, Stacy Wieseler, Lawrence M. Schopfer, Oksana Lockridge, Patrick Masson

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

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 122GFYS125 peptide segment, resulting in 122HFQT125. 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 kcat/Km 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.

Original languageEnglish (US)
Pages (from-to)1470-1478
Number of pages9
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1764
Issue number9
DOIs
StatePublished - Sep 2006

Keywords

  • Acetylcholinesterase
  • Molecular modelling
  • Organophosphate hydrolase activity
  • Organophosphorus inhibitors
  • Site-directed mutagenesis
  • Slow-binding inhibition

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

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