Effects of mutations of active site residues and amino acids interacting with the Ω loop on substrate activation of butyrylcholinesterase

Patrick Masson, Weihua Xie, Marie Thérèse Froment, Oksana Lockridge

Research output: Contribution to journalArticlepeer-review

43 Scopus citations


The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Ω loop. To determine whether the Ω loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10 000-fold, indicating severe alteration in the binding area in W430A. The kcat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Ω loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.

Original languageEnglish (US)
Pages (from-to)166-176
Number of pages11
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Issue number1-2
StatePublished - Jan 12 2001


  • Active site
  • Butyrylcholinesterase
  • Mutagenesis
  • Substrate activation
  • Ω loop

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology


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