Successive organophosphate inhibition and oxime reactivation reveals distinct responses of recombinant human cholinesterase variants
Identifieur interne : 002928 ( Main/Exploration ); précédent : 002927; suivant : 002929Successive organophosphate inhibition and oxime reactivation reveals distinct responses of recombinant human cholinesterase variants
Auteurs : Mikael Schwarz [Israël, États-Unis] ; Yael Loewenstein-Lichtenstein [Israël] ; David Glick [Israël] ; Jian Liao [Israël, États-Unis] ; Bent Norgaard-Pedersen [Danemark] ; Hermona Soreq [Israël]Source :
- Molecular Brain Research [ 0169-328X ] ; 1995.
Abstract
To explore the molecular basis of the biochemical differences among acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and their alternative splicing and allelic variants, we investigated the acylation phase of cholinesterase catalysis, using phosphorylation as an analogous reaction. Rate constants for organophosphate (DFP) inactivation, as well as for oxime (PAM)-promoted reactivation, were calculated for antibody-immobilized human cholinesterases produced in Xenopus oocytes from natural and site-directed variants of the corresponding DNA constructs. BuChE displayed inactivation and reactivation rates 200- and 25-fold higher than either product of 3′-variable ACNE DNAs, consistent with a putative in vivo function for BuChE as a detoxifier that protects AChE from inactivation. Chimeric substitution of active site gorge-lining residues in BuChE with the more anionic and aromatic residues of AChE, reduced inactivation 60-fold but reactivation only 4-fold, and the rate-limiting step of its catalysis appeared to be deacylation. In contrast, a positive charge at the acyl-binding site of BuChE decreased inactivation 8-fold and reactivation 30-fold. Finally, substitution of Asp70 by glycine, as in the natural ‘atypical’ BuChE variant, did not change the inactivation rate yet reduced reactivation 4-fold. Thus, a combination of electrostatic active site charges with aromatic residue differences at the gorge lining can explain the biochemical distinction between AChE and BuChE. Also, gorge-lining residues, including Asp70, appear to affect the deacylation step of catalysis by BuChE. Individuals carrying the ‘atypical’ BuChE allele may hence be unresponsive to oxime reactivation therapy following organophosphate poisoning.
Url:
DOI: 10.1016/0169-328X(95)00040-Y
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organophosphate inhibition and oxime reactivation reveals distinct responses of recombinant human cholinesterase variants</title><author><name sortKey="Schwarz, Mikael" sort="Schwarz, Mikael" uniqKey="Schwarz M" first="Mikael" last="Schwarz">Mikael Schwarz</name><affiliation wicri:level="1"><country xml:lang="fr">Israël</country><wicri:regionArea>Department of Biological Chemistry, The Life Sciences Institute, The Hebrew University of Jerusalem, 91904 Jerusalem</wicri:regionArea><wicri:noRegion>91904 Jerusalem</wicri:noRegion></affiliation><affiliation wicri:level="2"><country xml:lang="fr" wicri:curation="lc">États-Unis</country><wicri:regionArea>1 Present address: Department of Cell Biology, Stanford University School of Medicine, Stanford, CA 94305</wicri:regionArea><placeName><region type="state">Californie</region></placeName></affiliation></author><author><name sortKey="Loewenstein Lichtenstein, Yael" sort="Loewenstein Lichtenstein, Yael" uniqKey="Loewenstein Lichtenstein Y" 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Biological Chemistry, The Life Sciences Institute, The Hebrew University of Jerusalem, 91904 Jerusalem</wicri:regionArea><wicri:noRegion>91904 Jerusalem</wicri:noRegion></affiliation><affiliation wicri:level="2"><country xml:lang="fr" wicri:curation="lc">États-Unis</country><wicri:regionArea>2 Present address: Division of Gastroenterology, Stanford University School of Medicine, Stanford, CA 94305</wicri:regionArea><placeName><region type="state">Californie</region></placeName></affiliation></author><author><name sortKey="Norgaard Pedersen, Bent" sort="Norgaard Pedersen, Bent" uniqKey="Norgaard Pedersen B" first="Bent" last="Norgaard-Pedersen">Bent Norgaard-Pedersen</name><affiliation wicri:level="1"><country xml:lang="fr">Danemark</country><wicri:regionArea>Statens Seruminstitut, 2300 Copenhagen</wicri:regionArea><placeName><settlement type="city">Copenhague</settlement><region type="région" nuts="2">Hovedstaden</region></placeName></affiliation></author><author><name sortKey="Soreq, Hermona" sort="Soreq, Hermona" uniqKey="Soreq H" first="Hermona" last="Soreq">Hermona Soreq</name><affiliation><wicri:noCountry code="no comma">Corresponding author. Fax: (972) (2) 652-0258.</wicri:noCountry></affiliation><affiliation wicri:level="1"><country xml:lang="fr">Israël</country><wicri:regionArea>Department of Biological Chemistry, The Life Sciences Institute, The Hebrew University of Jerusalem, 91904 Jerusalem</wicri:regionArea><wicri:noRegion>91904 Jerusalem</wicri:noRegion></affiliation></author></analytic><monogr></monogr><series><title level="j">Molecular Brain Research</title><title level="j" type="abbrev">BRESM</title><idno type="ISSN">0169-328X</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1995">1995</date><biblScope unit="volume">31</biblScope><biblScope unit="issue">1–2</biblScope><biblScope unit="page" from="101">101</biblScope><biblScope unit="page" to="110">110</biblScope></imprint><idno type="ISSN">0169-328X</idno></series><idno type="istex">1BF798A24D405E81671996461FC1EF731F5CBB51</idno><idno type="DOI">10.1016/0169-328X(95)00040-Y</idno><idno type="PII">0169-328X(95)00040-Y</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0169-328X</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">To explore the molecular basis of the biochemical differences among acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and their alternative splicing and allelic variants, we investigated the acylation phase of cholinesterase catalysis, using phosphorylation as an analogous reaction. Rate constants for organophosphate (DFP) inactivation, as well as for oxime (PAM)-promoted reactivation, were calculated for antibody-immobilized human cholinesterases produced in Xenopus oocytes from natural and site-directed variants of the corresponding DNA constructs. BuChE displayed inactivation and reactivation rates 200- and 25-fold higher than either product of 3′-variable ACNE DNAs, consistent with a putative in vivo function for BuChE as a detoxifier that protects AChE from inactivation. Chimeric substitution of active site gorge-lining residues in BuChE with the more anionic and aromatic residues of AChE, reduced inactivation 60-fold but reactivation only 4-fold, and the rate-limiting step of its catalysis appeared to be deacylation. In contrast, a positive charge at the acyl-binding site of BuChE decreased inactivation 8-fold and reactivation 30-fold. Finally, substitution of Asp70 by glycine, as in the natural ‘atypical’ BuChE variant, did not change the inactivation rate yet reduced reactivation 4-fold. Thus, a combination of electrostatic active site charges with aromatic residue differences at the gorge lining can explain the biochemical distinction between AChE and BuChE. Also, gorge-lining residues, including Asp70, appear to affect the deacylation step of catalysis by BuChE. Individuals carrying the ‘atypical’ BuChE allele may hence be unresponsive to oxime reactivation therapy following organophosphate poisoning.</div></front></TEI><affiliations><list><country><li>Danemark</li><li>Israël</li><li>États-Unis</li></country><region><li>Californie</li><li>Hovedstaden</li></region><settlement><li>Copenhague</li></settlement></list><tree><country name="Israël"><noRegion><name sortKey="Schwarz, Mikael" sort="Schwarz, Mikael" uniqKey="Schwarz M" first="Mikael" last="Schwarz">Mikael Schwarz</name></noRegion><name sortKey="Glick, David" sort="Glick, David" uniqKey="Glick D" first="David" last="Glick">David Glick</name><name sortKey="Liao, Jian" sort="Liao, Jian" uniqKey="Liao J" first="Jian" last="Liao">Jian Liao</name><name sortKey="Loewenstein Lichtenstein, Yael" sort="Loewenstein Lichtenstein, Yael" uniqKey="Loewenstein Lichtenstein Y" first="Yael" last="Loewenstein-Lichtenstein">Yael Loewenstein-Lichtenstein</name><name sortKey="Soreq, Hermona" sort="Soreq, Hermona" uniqKey="Soreq H" first="Hermona" last="Soreq">Hermona Soreq</name></country><country name="États-Unis"><region name="Californie"><name sortKey="Schwarz, Mikael" sort="Schwarz, Mikael" uniqKey="Schwarz M" first="Mikael" last="Schwarz">Mikael Schwarz</name></region><name sortKey="Liao, Jian" sort="Liao, Jian" uniqKey="Liao J" first="Jian" last="Liao">Jian Liao</name></country><country name="Danemark"><region name="Hovedstaden"><name sortKey="Norgaard Pedersen, Bent" sort="Norgaard Pedersen, Bent" uniqKey="Norgaard Pedersen B" first="Bent" last="Norgaard-Pedersen">Bent Norgaard-Pedersen</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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