Butyrylcholinesterase-catalyzed hydrolysis of N-methylindoxyl acetate: Analysis of volume changes upon reaction and hysteretic behavior

Patrick Masson, Marie Thérèse Froment, Sébastien Fort, Fabien Ribes, Nicole Bec, Claude Balny, Lawrence M. Schopfer

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Hydrolysis of the neutral substrate N-methylindoxyl acetate (NMIA) by wild-type human butyrylcholinesterase (BuChE) and peripheral site mutants (D70G, Y332A, D70G/Y332A) was found to follow the Michaelis-Menten kinetics. Km was 0.14 mM for wild-type, and 0.07-0.16 mM for D70G, Y332A and D70G/Y332A, indicating that the peripheral site is not involved in NMIA binding. The values of kcat were of the same order for all enzymes: 12,000-18,000 min-1. Volume changes upon substrate binding (-ΔVKm) and the activation volumes (ΔVkcat) associated with hydrolysis of NMIA were calculated from the pressure dependence of the catalytic constants. Values of -ΔVKm indicate that NMIA binds to an aromatic residue, presumed to be W82, the active site binding locus. Binding is accompanied by a release of water molecules from the gorge. Residue 70 controls the number of water molecules that are released upon substrate binding. The values of ΔVkcat, which are positive for wild-type and faintly positive for D70G, clearly indicate that the catalytic steps are accompanied by re-entry of water into the gorge. Results support the premise that residue D70 is involved in the conformational stabilization of the active site gorge and in control of its hydration. A slow transient, preceding the steady state, was seen on a time scale of several minutes. The induction time rapidly increased with NMIA concentration to reach a limit at substrate saturation. Much shorter induction times (<1 min) were seen for hydrolysis of benzoylcholine (BzCh) by wild-type BuChE and for hydrolysis of butyrylthiocholine (BuSCh) by the active site mutants E197Q and E197Q/G117H. This slow transient was interpreted in terms of hysteresis without kinetic cooperativity. The hysteretic behavior of BuChE results from a slow conformational equilibrium between two enzyme states E and E′. NMIA binds only to the primed form E′. Kosmotropic salts and hydrostatic pressure were found to shift the equilibrium toward E′. The E→E′ transition is accompanied by a negative activation volume (ΔV0=-45±10 ml/mol), and the E′ form is more compact than E. Hydration water in the gorge of E′ appears to be more structured than in the unprimed form.

Original languageEnglish (US)
Pages (from-to)229-243
Number of pages15
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Issue number2
StatePublished - Jun 3 2002


  • Butyrylcholinesterase
  • Double-mutant cycle
  • Hydrostatic pressure
  • Hysteresis
  • Lyotropic salt
  • Slow conformational change
  • Transient
  • Volume change

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology


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