Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate

Patrick Masson, Boris N. Goldstein, Jean Claude Debouzy, Marie Thérèse Froment, Oksana Lockridge, Lawrence M Schopfer

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Steady-state kinetics for the hydrolysis of benzoylcholine (BzCh) and benzoylthiocholine (BzSCh) by wild-type human butyrylcholinesterase (BuChE) and by the peripheral anionic site mutant D70G were compared. kcat/K m for the hydrolysis of BzSCh was 17-fold and 32-fold lower than that for hydrolysis of BzCh by wild-type and D70G, respectively. The rate-limiting step for hydrolysis of BzCh was deacylation, whereas acylation was rate-limiting for hydrolysis of BzSCh. Wild-type enzyme and the D70G mutant were found to reach steady-state velocity slowly with BzCh as the substrate. At pH 6, the approach to steady-state for both enzymes consisted of a mono-exponential acceleration upon which a set of damped oscillations was superimposed. From pH 7 to 8.5, the approach to steady-state consisted of a simple exponential acceleration. The damped oscillations were analyzed by both a numerical approximation and simulation based on a theoretical model. BuChE-catalyzed hydrolysis of the thiocholine analogue of BzCh showed neither lags nor oscillations, under the same conditions. The frequency and amplitude of the damped oscillations decreased as the BzCh concentration increased. The apparent induction time for the exponential portion of the lag was calculated from the envelope of the damped oscillations or from the smooth lag. Wild-type BuChE showed a hyperbolic increase in induction time as the BzCh concentration increased (τmax = 210 s at pH 6.0). However, the induction time for D70G was constant over the whole range of BzCh concentrations (τ max = 60 s at pH 6.0). Thus, the induction time does not conform to a simple hysteretic model in which there is a slow conformational transition of the enzyme from an inactive form E to an active form E′. No pH-dependence of the induction time was found between pH 6.0 and 8.5 in sodium phosphate buffers of various concentrations (from 1 mM to 1 M). However, increasing the pH tended to abolish the oscillations (increase the damping factor). This effect was more pronounced for D70G than for wild-type. Although the lyotropic properties of phosphate change from chaotropic at pH 6.0 to kosmotropic at pH > 8.0, no effect of phosphate concentration on the oscillations was noticed at the different pH values, suggesting that the oscillations are not related to a pH-dependent Hofmeister effect of phosphate ions. Simulation and theoretical analysis of the oscillatory behaviour of the approach to the steady-state for BuChE led us to propose a model for the hysteresis of BuChE with BzCh. In this model, the substrate-free enzyme is present as an equilibrium mixture of two forms, E and E′. Substrate binds to E and E′, but only E′S makes products. It is proposed that oscillations originate from a time-dependent change in the local concentration, solvation and/or conformation of substrate in the bulk solution. 1H-NMR measurements provided evidence for a slow equilibrium between two BzCh conformers. Binding of the conformationally preferred substrate conformer leads to products.

Original languageEnglish (US)
Pages (from-to)220-234
Number of pages15
JournalEuropean Journal of Biochemistry
Volume271
Issue number1
DOIs
StatePublished - Jan 1 2004

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Benzoylcholine
Butyrylcholinesterase
Substrates
Hydrolysis
Phosphates
Enzymes
Thiocholine
Acylation
Solvation

Keywords

  • Benzoylcholine
  • Butyrylcholinesterase
  • Damped oscillations
  • Hysteresis
  • Slow conformational change

ASJC Scopus subject areas

  • Biochemistry

Cite this

Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate. / Masson, Patrick; Goldstein, Boris N.; Debouzy, Jean Claude; Froment, Marie Thérèse; Lockridge, Oksana; Schopfer, Lawrence M.

In: European Journal of Biochemistry, Vol. 271, No. 1, 01.01.2004, p. 220-234.

Research output: Contribution to journalArticle

Masson, Patrick ; Goldstein, Boris N. ; Debouzy, Jean Claude ; Froment, Marie Thérèse ; Lockridge, Oksana ; Schopfer, Lawrence M. / Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate. In: European Journal of Biochemistry. 2004 ; Vol. 271, No. 1. pp. 220-234.
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T1 - Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate

AU - Masson, Patrick

AU - Goldstein, Boris N.

AU - Debouzy, Jean Claude

AU - Froment, Marie Thérèse

AU - Lockridge, Oksana

AU - Schopfer, Lawrence M

PY - 2004/1/1

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N2 - Steady-state kinetics for the hydrolysis of benzoylcholine (BzCh) and benzoylthiocholine (BzSCh) by wild-type human butyrylcholinesterase (BuChE) and by the peripheral anionic site mutant D70G were compared. kcat/K m for the hydrolysis of BzSCh was 17-fold and 32-fold lower than that for hydrolysis of BzCh by wild-type and D70G, respectively. The rate-limiting step for hydrolysis of BzCh was deacylation, whereas acylation was rate-limiting for hydrolysis of BzSCh. Wild-type enzyme and the D70G mutant were found to reach steady-state velocity slowly with BzCh as the substrate. At pH 6, the approach to steady-state for both enzymes consisted of a mono-exponential acceleration upon which a set of damped oscillations was superimposed. From pH 7 to 8.5, the approach to steady-state consisted of a simple exponential acceleration. The damped oscillations were analyzed by both a numerical approximation and simulation based on a theoretical model. BuChE-catalyzed hydrolysis of the thiocholine analogue of BzCh showed neither lags nor oscillations, under the same conditions. The frequency and amplitude of the damped oscillations decreased as the BzCh concentration increased. The apparent induction time for the exponential portion of the lag was calculated from the envelope of the damped oscillations or from the smooth lag. Wild-type BuChE showed a hyperbolic increase in induction time as the BzCh concentration increased (τmax = 210 s at pH 6.0). However, the induction time for D70G was constant over the whole range of BzCh concentrations (τ max = 60 s at pH 6.0). Thus, the induction time does not conform to a simple hysteretic model in which there is a slow conformational transition of the enzyme from an inactive form E to an active form E′. No pH-dependence of the induction time was found between pH 6.0 and 8.5 in sodium phosphate buffers of various concentrations (from 1 mM to 1 M). However, increasing the pH tended to abolish the oscillations (increase the damping factor). This effect was more pronounced for D70G than for wild-type. Although the lyotropic properties of phosphate change from chaotropic at pH 6.0 to kosmotropic at pH > 8.0, no effect of phosphate concentration on the oscillations was noticed at the different pH values, suggesting that the oscillations are not related to a pH-dependent Hofmeister effect of phosphate ions. Simulation and theoretical analysis of the oscillatory behaviour of the approach to the steady-state for BuChE led us to propose a model for the hysteresis of BuChE with BzCh. In this model, the substrate-free enzyme is present as an equilibrium mixture of two forms, E and E′. Substrate binds to E and E′, but only E′S makes products. It is proposed that oscillations originate from a time-dependent change in the local concentration, solvation and/or conformation of substrate in the bulk solution. 1H-NMR measurements provided evidence for a slow equilibrium between two BzCh conformers. Binding of the conformationally preferred substrate conformer leads to products.

AB - Steady-state kinetics for the hydrolysis of benzoylcholine (BzCh) and benzoylthiocholine (BzSCh) by wild-type human butyrylcholinesterase (BuChE) and by the peripheral anionic site mutant D70G were compared. kcat/K m for the hydrolysis of BzSCh was 17-fold and 32-fold lower than that for hydrolysis of BzCh by wild-type and D70G, respectively. The rate-limiting step for hydrolysis of BzCh was deacylation, whereas acylation was rate-limiting for hydrolysis of BzSCh. Wild-type enzyme and the D70G mutant were found to reach steady-state velocity slowly with BzCh as the substrate. At pH 6, the approach to steady-state for both enzymes consisted of a mono-exponential acceleration upon which a set of damped oscillations was superimposed. From pH 7 to 8.5, the approach to steady-state consisted of a simple exponential acceleration. The damped oscillations were analyzed by both a numerical approximation and simulation based on a theoretical model. BuChE-catalyzed hydrolysis of the thiocholine analogue of BzCh showed neither lags nor oscillations, under the same conditions. The frequency and amplitude of the damped oscillations decreased as the BzCh concentration increased. The apparent induction time for the exponential portion of the lag was calculated from the envelope of the damped oscillations or from the smooth lag. Wild-type BuChE showed a hyperbolic increase in induction time as the BzCh concentration increased (τmax = 210 s at pH 6.0). However, the induction time for D70G was constant over the whole range of BzCh concentrations (τ max = 60 s at pH 6.0). Thus, the induction time does not conform to a simple hysteretic model in which there is a slow conformational transition of the enzyme from an inactive form E to an active form E′. No pH-dependence of the induction time was found between pH 6.0 and 8.5 in sodium phosphate buffers of various concentrations (from 1 mM to 1 M). However, increasing the pH tended to abolish the oscillations (increase the damping factor). This effect was more pronounced for D70G than for wild-type. Although the lyotropic properties of phosphate change from chaotropic at pH 6.0 to kosmotropic at pH > 8.0, no effect of phosphate concentration on the oscillations was noticed at the different pH values, suggesting that the oscillations are not related to a pH-dependent Hofmeister effect of phosphate ions. Simulation and theoretical analysis of the oscillatory behaviour of the approach to the steady-state for BuChE led us to propose a model for the hysteresis of BuChE with BzCh. In this model, the substrate-free enzyme is present as an equilibrium mixture of two forms, E and E′. Substrate binds to E and E′, but only E′S makes products. It is proposed that oscillations originate from a time-dependent change in the local concentration, solvation and/or conformation of substrate in the bulk solution. 1H-NMR measurements provided evidence for a slow equilibrium between two BzCh conformers. Binding of the conformationally preferred substrate conformer leads to products.

KW - Benzoylcholine

KW - Butyrylcholinesterase

KW - Damped oscillations

KW - Hysteresis

KW - Slow conformational change

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