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

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

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
Volume1597
Issue number2
DOIs
StatePublished - Jun 3 2002

Fingerprint

Butyrylcholinesterase
Hydrolysis
Catalytic Domain
Water
Substrates
Hydration
Benzoylcholine
Butyrylthiocholine
Chemical activation
Molecules
Hydrostatic Pressure
Kinetics
Reentry
Hydrostatic pressure
Enzymes
Hysteresis
N-methylindoxyl acetate
Stabilization
Salts
Binding Sites

Keywords

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

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Butyrylcholinesterase-catalyzed hydrolysis of N-methylindoxyl acetate : Analysis of volume changes upon reaction and hysteretic behavior. / Masson, Patrick; Froment, Marie Thérèse; Fort, Sébastien; Ribes, Fabien; Bec, Nicole; Balny, Claude; Schopfer, Lawrence M.

In: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, Vol. 1597, No. 2, 03.06.2002, p. 229-243.

Research output: Contribution to journalArticle

Masson, Patrick ; Froment, Marie Thérèse ; Fort, Sébastien ; Ribes, Fabien ; Bec, Nicole ; Balny, Claude ; Schopfer, Lawrence M. / Butyrylcholinesterase-catalyzed hydrolysis of N-methylindoxyl acetate : Analysis of volume changes upon reaction and hysteretic behavior. In: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology. 2002 ; Vol. 1597, No. 2. pp. 229-243.
@article{9c540a5ff6c7412b8d0a25fbbc620ce4,
title = "Butyrylcholinesterase-catalyzed hydrolysis of N-methylindoxyl acetate: Analysis of volume changes upon reaction and hysteretic behavior",
abstract = "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.",
keywords = "Butyrylcholinesterase, Double-mutant cycle, Hydrostatic pressure, Hysteresis, Lyotropic salt, Slow conformational change, Transient, Volume change",
author = "Patrick Masson and Froment, {Marie Th{\'e}r{\`e}se} and S{\'e}bastien Fort and Fabien Ribes and Nicole Bec and Claude Balny and Schopfer, {Lawrence M}",
year = "2002",
month = "6",
day = "3",
doi = "10.1016/S0167-4838(02)00265-0",
language = "English (US)",
volume = "1597",
pages = "229--243",
journal = "Biochimica et Biophysica Acta - Proteins and Proteomics",
issn = "1570-9639",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Butyrylcholinesterase-catalyzed hydrolysis of N-methylindoxyl acetate

T2 - Analysis of volume changes upon reaction and hysteretic behavior

AU - Masson, Patrick

AU - Froment, Marie Thérèse

AU - Fort, Sébastien

AU - Ribes, Fabien

AU - Bec, Nicole

AU - Balny, Claude

AU - Schopfer, Lawrence M

PY - 2002/6/3

Y1 - 2002/6/3

N2 - 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.

AB - 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.

KW - Butyrylcholinesterase

KW - Double-mutant cycle

KW - Hydrostatic pressure

KW - Hysteresis

KW - Lyotropic salt

KW - Slow conformational change

KW - Transient

KW - Volume change

UR - http://www.scopus.com/inward/record.url?scp=0037013985&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037013985&partnerID=8YFLogxK

U2 - 10.1016/S0167-4838(02)00265-0

DO - 10.1016/S0167-4838(02)00265-0

M3 - Article

VL - 1597

SP - 229

EP - 243

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 2

ER -