Structural approach to the aging of phosphylated cholinesterases

Patrick Masson, Florian Nachon, Oksana Lockridge

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

Phosphylated cholinesterases (ChE) can undergo a side reaction that progressively decreases their reactivatability. This process, termed " aging" , results from dealkylation of the adduct and depends on the structure of the organophosphyl moiety. Aged ChEs are resistant to reactivation by oximes.Owing to the toxicological importance of OPs, the molecular mechanism of aging has been the subject of research for decades. It was not clear whether aging involves the same bond breakage regardless the type of OP or is a scission of P-O-C bonds (P-O or O-C) in phosphates/phosphonates, P-N-C bonds in phosphoramidates, and P-S-C bonds in phosphonothionates. It was assumed that the resulting negatively charged atom on phosphorus of the aged adduct prevented nucleophilic attack by oximates, but studies on negatively charged model molecules do not support this hypothesis. Decrease in conformational flexibility of aged enzymes may contribute to their non-reactivatability by preventing proper adjustment of reactivators in the active site gorge.MALDI-TOF mass spectrometry of phosphylated human butyrylcholinesterase (hBChE) in water and in 18O-water provided evidence that aging results from O-C breakage, i.e. O-dealkylation. In contrast, the isomalathion-BChE conjugate ages mostly through P-S bond cleavage, but a minor product results from O-C and/or S-C breakage. The crystal structures of hBChE and hAChE inhibited by tabun showed that aging of tabun-ChE conjugates results from O-dealkylation. However, depending on the nature of O-alkyl and N-alkyl chains, aging of BChE inhibited by other phosphoramidates results either from O-C breakage or deamination, i.e. P-N breakage. It was found that dealkylation of branched alkoxy involves a transient carbocation. Dealkylation of OP-ChE conjugates is accompanied by enzyme conformational changes. Urea, organic solvent, heat and pressure denaturation of human BChE showed that the conformational stability of aged OP-BChE conjugates is dramatically increased compared to native enzyme. Determination of the three-dimensional structure of BChE and AChE conjugated to different OPs showed that aged adducts form a salt bridge with the protonated catalytic histidine. Structure alteration of aged enzymes is accompanied by exit of water molecules from the enzyme's active site gorge. In addition, neutron scattering studies provided evidence that the structural dynamics of aged BChE is dramatically altered compared to native enzyme. Knowledge of the molecular basis of aging will help to design reactivators of aged ChEs, molecules capable of slowing the aging process, and pseudocatalytic ChE-based bioscavengers.

Original languageEnglish (US)
Pages (from-to)157-162
Number of pages6
JournalChemico-Biological Interactions
Volume187
Issue number1-3
DOIs
StatePublished - Sep 1 2010

Fingerprint

Dealkylation
Cholinesterases
Aging of materials
Enzymes
Butyrylcholinesterase
Water
Catalytic Domain
Molecules
Organophosphonates
Deamination
Oximes
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Neutrons
Histidine
Toxicology
Phosphorus
Denaturation
Urea
Mass Spectrometry
Structural dynamics

Keywords

  • Aging
  • Cholinesterase
  • Organophosphorus compounds

ASJC Scopus subject areas

  • Toxicology

Cite this

Structural approach to the aging of phosphylated cholinesterases. / Masson, Patrick; Nachon, Florian; Lockridge, Oksana.

In: Chemico-Biological Interactions, Vol. 187, No. 1-3, 01.09.2010, p. 157-162.

Research output: Contribution to journalArticle

Masson, Patrick ; Nachon, Florian ; Lockridge, Oksana. / Structural approach to the aging of phosphylated cholinesterases. In: Chemico-Biological Interactions. 2010 ; Vol. 187, No. 1-3. pp. 157-162.
@article{ed5f0bd729834d5da4452d78e37ab387,
title = "Structural approach to the aging of phosphylated cholinesterases",
abstract = "Phosphylated cholinesterases (ChE) can undergo a side reaction that progressively decreases their reactivatability. This process, termed {"} aging{"} , results from dealkylation of the adduct and depends on the structure of the organophosphyl moiety. Aged ChEs are resistant to reactivation by oximes.Owing to the toxicological importance of OPs, the molecular mechanism of aging has been the subject of research for decades. It was not clear whether aging involves the same bond breakage regardless the type of OP or is a scission of P-O-C bonds (P-O or O-C) in phosphates/phosphonates, P-N-C bonds in phosphoramidates, and P-S-C bonds in phosphonothionates. It was assumed that the resulting negatively charged atom on phosphorus of the aged adduct prevented nucleophilic attack by oximates, but studies on negatively charged model molecules do not support this hypothesis. Decrease in conformational flexibility of aged enzymes may contribute to their non-reactivatability by preventing proper adjustment of reactivators in the active site gorge.MALDI-TOF mass spectrometry of phosphylated human butyrylcholinesterase (hBChE) in water and in 18O-water provided evidence that aging results from O-C breakage, i.e. O-dealkylation. In contrast, the isomalathion-BChE conjugate ages mostly through P-S bond cleavage, but a minor product results from O-C and/or S-C breakage. The crystal structures of hBChE and hAChE inhibited by tabun showed that aging of tabun-ChE conjugates results from O-dealkylation. However, depending on the nature of O-alkyl and N-alkyl chains, aging of BChE inhibited by other phosphoramidates results either from O-C breakage or deamination, i.e. P-N breakage. It was found that dealkylation of branched alkoxy involves a transient carbocation. Dealkylation of OP-ChE conjugates is accompanied by enzyme conformational changes. Urea, organic solvent, heat and pressure denaturation of human BChE showed that the conformational stability of aged OP-BChE conjugates is dramatically increased compared to native enzyme. Determination of the three-dimensional structure of BChE and AChE conjugated to different OPs showed that aged adducts form a salt bridge with the protonated catalytic histidine. Structure alteration of aged enzymes is accompanied by exit of water molecules from the enzyme's active site gorge. In addition, neutron scattering studies provided evidence that the structural dynamics of aged BChE is dramatically altered compared to native enzyme. Knowledge of the molecular basis of aging will help to design reactivators of aged ChEs, molecules capable of slowing the aging process, and pseudocatalytic ChE-based bioscavengers.",
keywords = "Aging, Cholinesterase, Organophosphorus compounds",
author = "Patrick Masson and Florian Nachon and Oksana Lockridge",
year = "2010",
month = "9",
day = "1",
doi = "10.1016/j.cbi.2010.03.027",
language = "English (US)",
volume = "187",
pages = "157--162",
journal = "Chemico-Biological Interactions",
issn = "0009-2797",
publisher = "Elsevier Ireland Ltd",
number = "1-3",

}

TY - JOUR

T1 - Structural approach to the aging of phosphylated cholinesterases

AU - Masson, Patrick

AU - Nachon, Florian

AU - Lockridge, Oksana

PY - 2010/9/1

Y1 - 2010/9/1

N2 - Phosphylated cholinesterases (ChE) can undergo a side reaction that progressively decreases their reactivatability. This process, termed " aging" , results from dealkylation of the adduct and depends on the structure of the organophosphyl moiety. Aged ChEs are resistant to reactivation by oximes.Owing to the toxicological importance of OPs, the molecular mechanism of aging has been the subject of research for decades. It was not clear whether aging involves the same bond breakage regardless the type of OP or is a scission of P-O-C bonds (P-O or O-C) in phosphates/phosphonates, P-N-C bonds in phosphoramidates, and P-S-C bonds in phosphonothionates. It was assumed that the resulting negatively charged atom on phosphorus of the aged adduct prevented nucleophilic attack by oximates, but studies on negatively charged model molecules do not support this hypothesis. Decrease in conformational flexibility of aged enzymes may contribute to their non-reactivatability by preventing proper adjustment of reactivators in the active site gorge.MALDI-TOF mass spectrometry of phosphylated human butyrylcholinesterase (hBChE) in water and in 18O-water provided evidence that aging results from O-C breakage, i.e. O-dealkylation. In contrast, the isomalathion-BChE conjugate ages mostly through P-S bond cleavage, but a minor product results from O-C and/or S-C breakage. The crystal structures of hBChE and hAChE inhibited by tabun showed that aging of tabun-ChE conjugates results from O-dealkylation. However, depending on the nature of O-alkyl and N-alkyl chains, aging of BChE inhibited by other phosphoramidates results either from O-C breakage or deamination, i.e. P-N breakage. It was found that dealkylation of branched alkoxy involves a transient carbocation. Dealkylation of OP-ChE conjugates is accompanied by enzyme conformational changes. Urea, organic solvent, heat and pressure denaturation of human BChE showed that the conformational stability of aged OP-BChE conjugates is dramatically increased compared to native enzyme. Determination of the three-dimensional structure of BChE and AChE conjugated to different OPs showed that aged adducts form a salt bridge with the protonated catalytic histidine. Structure alteration of aged enzymes is accompanied by exit of water molecules from the enzyme's active site gorge. In addition, neutron scattering studies provided evidence that the structural dynamics of aged BChE is dramatically altered compared to native enzyme. Knowledge of the molecular basis of aging will help to design reactivators of aged ChEs, molecules capable of slowing the aging process, and pseudocatalytic ChE-based bioscavengers.

AB - Phosphylated cholinesterases (ChE) can undergo a side reaction that progressively decreases their reactivatability. This process, termed " aging" , results from dealkylation of the adduct and depends on the structure of the organophosphyl moiety. Aged ChEs are resistant to reactivation by oximes.Owing to the toxicological importance of OPs, the molecular mechanism of aging has been the subject of research for decades. It was not clear whether aging involves the same bond breakage regardless the type of OP or is a scission of P-O-C bonds (P-O or O-C) in phosphates/phosphonates, P-N-C bonds in phosphoramidates, and P-S-C bonds in phosphonothionates. It was assumed that the resulting negatively charged atom on phosphorus of the aged adduct prevented nucleophilic attack by oximates, but studies on negatively charged model molecules do not support this hypothesis. Decrease in conformational flexibility of aged enzymes may contribute to their non-reactivatability by preventing proper adjustment of reactivators in the active site gorge.MALDI-TOF mass spectrometry of phosphylated human butyrylcholinesterase (hBChE) in water and in 18O-water provided evidence that aging results from O-C breakage, i.e. O-dealkylation. In contrast, the isomalathion-BChE conjugate ages mostly through P-S bond cleavage, but a minor product results from O-C and/or S-C breakage. The crystal structures of hBChE and hAChE inhibited by tabun showed that aging of tabun-ChE conjugates results from O-dealkylation. However, depending on the nature of O-alkyl and N-alkyl chains, aging of BChE inhibited by other phosphoramidates results either from O-C breakage or deamination, i.e. P-N breakage. It was found that dealkylation of branched alkoxy involves a transient carbocation. Dealkylation of OP-ChE conjugates is accompanied by enzyme conformational changes. Urea, organic solvent, heat and pressure denaturation of human BChE showed that the conformational stability of aged OP-BChE conjugates is dramatically increased compared to native enzyme. Determination of the three-dimensional structure of BChE and AChE conjugated to different OPs showed that aged adducts form a salt bridge with the protonated catalytic histidine. Structure alteration of aged enzymes is accompanied by exit of water molecules from the enzyme's active site gorge. In addition, neutron scattering studies provided evidence that the structural dynamics of aged BChE is dramatically altered compared to native enzyme. Knowledge of the molecular basis of aging will help to design reactivators of aged ChEs, molecules capable of slowing the aging process, and pseudocatalytic ChE-based bioscavengers.

KW - Aging

KW - Cholinesterase

KW - Organophosphorus compounds

UR - http://www.scopus.com/inward/record.url?scp=77955513808&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77955513808&partnerID=8YFLogxK

U2 - 10.1016/j.cbi.2010.03.027

DO - 10.1016/j.cbi.2010.03.027

M3 - Article

VL - 187

SP - 157

EP - 162

JO - Chemico-Biological Interactions

JF - Chemico-Biological Interactions

SN - 0009-2797

IS - 1-3

ER -