Structural and hydration changes in the active site gorge of phosporhylated butyrylcholinesterase accompanying the aging process

Masson, Patrick; Fortier, Pierre Louis; Albaret, Christine; Cléry, Cécile; Guerra, Patrice; Lockridge, Oksana

doi:10.1016/s0009-2797(99)00010-1

Cited by 6 publications

(10 citation statements)

References 36 publications

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“…Thus, D70 plays a key role in the conformational stability of the BuChE active site gorge. This corroborates the conclusion that D70 is a major residue involved in the water/H‐bond network dynamics of the active site gorge of BuChE as probed by hydrostatic vs osmotic stress experiments [43].…”

Section: Resultssupporting

confidence: 87%

“…In order to gain an insight into the molecular bases of the break in Arrhenius and van’t Hoff plots of wild‐type BuChE, we studied the pressure and temperature effects on the catalytic properties of the D70G mutant because the D→G mutation changes both the affinity for positively charged substrates and the catalytic behaviour of the enzyme [5, 9, 13], and because residue D70 has been found to control the water/H‐bond network and flux of water molecules in the gorge [43]. Kinetic analysis of the mutant enzyme was carried out in the pressure range 0.001–1.5 kbar (at pressure higher than 1.5 kbar, the enzyme lost its activity below 25 °C).…”

Section: Resultsmentioning

confidence: 99%

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Differential effect of pressure and temperature on the catalytic behaviour of wild‐type human butyrylcholinesterase and its D70G mutant

Weingand-Ziadé

Renault

Masson

1999

European Journal of Biochemistry

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The combined action of temperature (10±358C) and pressure (0.001±2 kbar) on the catalytic activity of wild-type human butyrylcholinesterase (BuChE) and its D70G mutant was investigated at pH 7.0 using butyrylthiocholine as the substrate. The residue D70, located at the mouth of the active site gorge, is an essential component of the peripheral substrate binding site of BuChE. Results showed a break in Arrhenius plots of wild-type BuChE (at T t < 22 8C) whatever the pressure (dT t /dP = 1.6^1.5 8C´kbar 21 ), whereas no break was observed in Arrhenius plots of the D70G mutant. These results suggested a temperature-induced conformational change of the wild-type BuChE which did not occur for the D70G mutant. For the wild-type BuChE, at around a pressure of 1 kbar, an intermediate state, whose affinity for substrate was increased, appeared. This intermediate state was not seen for the mutant enzyme. The wild-type BuChE remained active up to a pressure of 2 kbar whatever the temperature, whereas the D70G mutant was found to be more sensitive to pressure inactivation (at pressures higher than 1.5 kbar the mutant enzyme lost its activity at temperatures lower than 25 8C). The results indicate that the residue D70 controls the conformational plasticity of the active site gorge of BuChE, and is involved in regulation of the catalytic activity as a function of temperature.

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Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Differential effect of pressure and temperature on the catalytic behaviour of wild‐type human butyrylcholinesterase and its D70G mutant

Weingand-Ziadé

Renault

Masson

1999

European Journal of Biochemistry

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“…Replacement of Glu199 resulted in a decrease in the rate of aging, however, the similar pH dependence of wild type and Glu199 mutant enzymes indicate that this residue is not involved in proton transfer [138]. The aging rate was also decreased when Asp72 was replaced with nonionic residues [34,119].…”

Section: Reactions With Pseudosubstrates and Irreversible Inhibitionmentioning

confidence: 93%

“…Some OP-enzyme adducts undergo a dealkylation reaction, which converts the enzyme into non-re-activatable species, resulting in truly irreversible inhibition of the enzyme. This process, called "aging", prevents treatment and exacerbates the effects of human intoxication by OP agents [2,[117][118][119].…”

Section: Reactions With Pseudosubstrates and Irreversible Inhibitionmentioning

confidence: 99%

“…Although there is evidence that the aging process is facilitated by intermolecular interactions of the phosphonyl moiety with structural elements of the AChE active center, the specific role of the enzyme residues in this process is not well understood. The role of active site gorge residues on the rate of aging of corresponding OP conjugates has been studied by site directed mutagenesis [34,119,[137][138][139]. The most dramatic resistance to aging was observed when the Trp84 is replaced by alanine [138].…”

Section: Reactions With Pseudosubstrates and Irreversible Inhibitionmentioning

confidence: 99%

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Acetylcholinesterase: Mechanism of Catalysis and Inhibition

Tõugu

2001

CMCCNSA

150

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Recent advances in the study of the catalytic properties of acetylcholinesterases have been reviewed. The main biological function of this enzyme is the fast termination of impulse transmission at cholinegric synapses by rapid hydrolysis of the neurotransmitter acetylcholine. Acetylcholinesterase has been often characterized as a perfect enzyme because its catalytic properties have been tuned to the highest possible limit. However, it seems paradoxical that the active site of this enzyme is buried deeply inside the enzyme molecule in the bottom of a narrow gorge restricting the traffic of substrates and products. The analysis of recent advances in enzymology and data on cholinesterase revealed rationality of this organization. The primary task of an enzyme catalyst is to lower the activation barrier for the chemical transformation of the substrate, and the catalytic power of the enzyme originates in polar solvation of the transition state by properly oriented dipoles inside the enzyme molecule. The active site gorge of acetylcholinesterase, containing multiple potential substrate binding areas, is responsible for "trapping" and delivery of substrate molecules to the active site. The phenomena of "allosteric modulation" and substrate inhibition arise as secondary effects of the presence of the narrow gorge lined with hydrophobic residues.

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Fundamental Reaction Mechanism for Cocaine Hydrolysis in Human Butyrylcholinesterase

Chen

Zheng²,

Landry³

2003

J. Am. Chem. Soc.

139

284

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Butyrylcholinesterase (BChE)-cocaine binding and the fundamental pathway for BChE-catalyzed hydrolysis of cocaine have been studied by molecular modeling, molecular dynamics (MD) simulations, and ab initio calculations. Modeling and simulations indicate that the structures of the prereactive BChE/substrate complexes for (-)-cocaine and (+)-cocaine are all similar to that of the corresponding prereactive BChE/butyrylcholine (BCh) complex. The overall binding of BChE with (-)-cocaine and (+)-cocaine is also similar to that proposed with butyrylthiocholine and succinyldithiocholine, i.e., (-)- or (+)-cocaine first slides down the substrate-binding gorge to bind to Trp-82 and stands vertically in the gorge between Asp-70 and Trp-82 (nonprereactive complex) and then rotates to a position in the catalytic site within a favorable distance for nucleophilic attack and hydrolysis by Ser-198 (prereactive complex). In the prereactive complex, cocaine lies horizontally at the bottom of the gorge. The fundamental catalytic hydrolysis pathway, consisting of acylation and deacylation stages similar to those for ester hydrolysis by other serine hydrolases, was proposed on the basis of the simulated prereactive complex and confirmed theoretically by ab initio reaction coordinate calculations. Both the acylation and deacylation follow a double-proton-transfer mechanism. The calculated energetic results show that within the chemical reaction process the highest energy barrier and Gibbs free energy barrier are all associated with the first step of deacylation. The calculated ratio of the rate constant (k(cat)) for the catalytic hydrolysis to that (k(0)) for the spontaneous hydrolysis is approximately 9.0 x 10(7). The estimated k(cat)/k(0) value of approximately 9.0 x 10(7) is in excellent agreement with the experimentally derived k(cat)/k(0) value of approximately 7.2 x 10(7) for (+)-cocaine, whereas it is approximately 2000 times larger than the experimentally derived k(cat)/k(0) value of approximately 4.4 x 10(4) for (-)-cocaine. All of the results suggest that the rate-determining step of the BChE-catalyzed hydrolysis of (+)-cocaine is the first step of deacylation, whereas for (-)-cocaine the change from the nonprereactive complex to the prereactive complex is rate-determining and has a Gibbs free energy barrier higher than that for the first step of deacylation by approximately 4 kcal/mol. A further analysis of the structural changes from the nonprereactive complex to the prereactive complex reveals specific amino acid residues hindering the structural changes, providing initial clues for the rational design of BChE mutants with improved catalytic activity for (-)-cocaine.

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Structural and hydration changes in the active site gorge of phosporhylated butyrylcholinesterase accompanying the aging process

Cited by 6 publications

References 36 publications

Differential effect of pressure and temperature on the catalytic behaviour of wild‐type human butyrylcholinesterase and its D70G mutant

Differential effect of pressure and temperature on the catalytic behaviour of wild‐type human butyrylcholinesterase and its D70G mutant

Acetylcholinesterase: Mechanism of Catalysis and Inhibition

Fundamental Reaction Mechanism for Cocaine Hydrolysis in Human Butyrylcholinesterase

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