X-ray crystallographic snapshots of reaction intermediates in the G117H mutant of human butyrylcholinesterase, a nerve agent target engineered into a catalytic bioscavenger

Florian Nachon, Eugenie Carletti, Marielle Wandhammer, Yvain Nicolet, Lawrence M Schopfer, Patrick Masson, Oksana Lockridge

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

OPs (organophosphylates) exert their acute toxicity through inhibition of acetylcholinesterase, by phosphylation of the catalytic serine residue. Engineering of human butyrylcholinesterase, by substitution of a histidine residue for the glycine residue at position 117, led to the creation of OP hydrolase activity. However, the lack of structural information and poor understanding of the hydrolytic mechanism of the G117H mutant has hampered further improvements in the catalytic activity. We have solved the crystallographic structure of the G117H mutant with a variety of ligands in its active site. A sulfate anion bound to the active site suggested the positioning for an OP prior to phosphylation. A fluoride anion was found in the active site when NaF was added to the crystallization buffer. In the fluoride complex, the imidazole ring from the His117 residue was substantially shifted, adopting a relaxed conformation probably close to that of the unliganded mutant enzyme. Additional X-ray structures were obtained from the transient covalent adducts formed upon reaction of the G117H mutant with the OPs echothiophate and VX [ethyl ({2-[bis-(propan-2-yl)amino]ethyl}sulfanyl](methyl)phosphinate]. The position of the His117 residue shifted in response to the introduction of these adducts, overlaying the phosphylserine residue. These structural data suggest that the dephosphylation mechanism involves either a substantial conformational change of the His117 residue or an adjacent nucleophilic substitution by water.

Original languageEnglish (US)
Pages (from-to)73-82
Number of pages10
JournalBiochemical Journal
Volume434
Issue number1
DOIs
StatePublished - Feb 15 2011

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Butyrylcholinesterase
Reaction intermediates
Fluorides
Anions
Catalytic Domain
Substitution reactions
X-Rays
X rays
Hydrolases
Acetylcholinesterase
Crystallization
Histidine
Glycine
Serine
Sulfates
Toxicity
Conformations
Catalyst activity
Buffers
Human Engineering

Keywords

  • Acetylcholinesterase
  • Bioscavenger
  • Butyrylcholinesterase
  • Organophosphorus
  • Protein engineering
  • Spontaneous reactivation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

X-ray crystallographic snapshots of reaction intermediates in the G117H mutant of human butyrylcholinesterase, a nerve agent target engineered into a catalytic bioscavenger. / Nachon, Florian; Carletti, Eugenie; Wandhammer, Marielle; Nicolet, Yvain; Schopfer, Lawrence M; Masson, Patrick; Lockridge, Oksana.

In: Biochemical Journal, Vol. 434, No. 1, 15.02.2011, p. 73-82.

Research output: Contribution to journalArticle

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