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Rudy J. Richardson - One of the best experts on this subject based on the ideXlab platform.
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inhibition of acetylcholinesterases by stereoisomeric organophosphorus compounds containing both thioester and p nitrophenyl leaving groups
Chemical Research in Toxicology, 2020Co-Authors: Todd T Talley, Rudy J. Richardson, Clifford E. Berkman, Chihkai Chao, Charles M. ThompsonAbstract:Studies with acetylcholinesterase (AChE) inhibited by organophosphorus (OP) compounds with two chiral centers can serve as models or surrogates for understanding the rate, orientation, and postinhibitory mechanisms by the nerve agent soman that possesses dual phosphorus and carbon chiral centers. In the current approach, stereoisomers of O-methyl, [S-(succinic acid, diethyl ester), O-(4-nitrophenyl) phosphorothiolate (MSNPs) were synthesized, and the inhibition, reactivation, and aging mechanisms were studied with electric eel AChE (eeAChE) and recombinant mouse brain AChE (rmAChE). The MSNP RPRC isomer was the strongest inhibitor of both eeAChE and rmAChE at 8- and 24-fold greater potency, respectively, than the weakest SPSC isomer. eeAChE inhibited by the RPRC- or RPSC-MSNP isomer underwent spontaneous reactivation ∼10- to 20-fold faster than the enzyme inhibited by SPRC- and SPSC-MSNP, and only 4% spontaneous reactivation was observed from the SPRC-eeAChE adduct. Using 2-pyridine aldoxime methiodide (2-PAM) or trimedoxime (TMB-4), eeAChE inhibited by RPRC- or SPRC-MSNP reactivated up to 90% and 3- to 4-fold faster than eeAChE inhibited by the RPSC- or SPSC-MSNP isomer. Spontaneous reactivation rates for rmAChE were 1.5- to 10-fold higher following inhibition by RPSC- and SPSC-MSNPs than inhibition by either RC isomer, a trend opposite to that found for eeAChE. Oxime reactivation of rmAChE following inhibition by RPRC- and SPRC-MSNPs was 2.5- to 5-fold faster than inhibition by RPSC- or SPSC-MSNPs. Due to structural similarities, MSNPs that phosphylate AChE with the loss of the p-nitrophenoxy (PNP) group form identical, nonreactivatable adducts to those formed from SP-Isomalathion; however, all the MSNP isomers inhibited AChE to form adducts that reactivated. Thus, MSNPs inactivate AChE via the ejection of either PNP or thiosuccinyl groups to form a combination of reactivatable and nonreactivatable adducts, and this differs from the mechanism of AChE inhibition by Isomalathion.
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stereoselective inactivation of torpedo californica acetylcholinesterase by Isomalathion inhibitory reactions with 1r and 1s isomers proceed by different mechanisms
Chemical Research in Toxicology, 2003Co-Authors: Jonathan A Doorn, Charles M. Thompson, Robert B Christner, Rudy J. RichardsonAbstract:The present study was undertaken to test the hypothesis that acetylcholinesterase (AChE) inhibition by Isomalathion stereoisomers proceeds with different primary leaving groups for (1R)- and (1S)-isomers. Consistent with results obtained with enzyme from other species, AChE from Torpedo californica (TcAChE) was stereoselectively inhibited by Isomalathion isomers with the (1R,3R)-isomer exhibiting greater potency than (1S,3S)-Isomalathion. TcAChE modified by (1R)-isomers readily reactivated in the presence of 2-pralidoxime methiodide (2-PAM), whereas enzyme inhibited by (1S)-Isomalathions was intractable toward reactivation. Computer-based molecular modeling showed that the ligand positioned as the primary leaving group was diethyl thiosuccinyl for (1R)-isomers and thiomethyl for (1S)-Isomalathions. Mass spectral analysis revealed that inhibition of TcAChE by (1R)-isomers resulted in an O,S-dimethyl phosphate adduct, as expected from expulsion of the diethyl thiosuccinyl ligand. In contrast, inactivation of the enzyme by (1S)-Isomalathions yielded an O-methyl phosphate adduct, consistent with initial loss of thiomethyl followed by displacement of the diethyl thiosuccinyl group. The findings demonstrate that the inhibitory reactions of TcAChE with (1R)- and (1S)-Isomalathions proceed by different mechanisms involving distinct primary leaving groups.
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identification of butyrylcholinesterase adducts after inhibition with Isomalathion using mass spectrometry difference in mechanism between 1r and 1s stereoisomers
Toxicology and Applied Pharmacology, 2001Co-Authors: Jonathan A Doorn, Charles M. Thompson, Todd T Talley, Michael Schall, Douglas A Gage, Rudy J. RichardsonAbstract:Previous kinetic studies found that butyrylcholinesterase (BChE) inhibited by (1R)-Isomalathions readily reactivated, while enzyme inactivated by (1S)-isomers did not. This study tested the hypothesis that (1R)- and (1S)-isomers inhibit BChE by different mechanisms, yielding distinct adducts identifiable by peptide mass mapping with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS). Equine BChE (EBChE) was inhibited to <10% of control activity with each isomer of Isomalathion and the reference compound isoparathion methyl. Control and treated enzyme was digested with trypsin, and peptides were fractionated with HPLC. Separated and unseparated peptides were analyzed with MALDI–TOF–MS. Identity of an organophosphorus peptide adduct was confirmed by fragmentation using postsource decay analysis. EBChE inhibited by (1R)-Isomalathions or (S)-isoparathion methyl readily reactivated after oxime treatment with 30–40% activity recovered. Enzyme inactivated by (1S)-Isomalathions or (R)-isoparathion methyl recovered <2% and <5% activity, respectively, after oxime treatment. MALDI–TOF–MS analysis revealed that inhibition of EBChE by (1R)-Isomalathions and (R)- or (S)-isoparathion methyl yielded O,S-dimethyl phosphate adducts. Enzyme inactivated by (1S)-Isomalathions produced only O-methyl phosphate adduct. EBChE modified by (1R)-Isomalathions or either enantiomer of isoparathion methyl yielded an O-methyl phosphate adduct as well. The results indicate that EBChE inhibition by (1R)-Isomalathions proceeds with loss of diethyl thiosuccinate, but inactivation by (1S)-isomers occurs with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate to yield an aged enzyme. Furthermore, the data suggest that aging of the O,S-dimethyl phosphate adduct occurs via an SN2 process with loss of thiomethyl.
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probing the active sites of butyrylcholinesterase and cholesterol esterase with Isomalathion conserved stereoselective inactivation of serine hydrolases structurally related to acetylcholinesterase
Chemical Research in Toxicology, 2001Co-Authors: Jonathan A Doorn, Todd T Talley, Charles M. Thompson, Rudy J. RichardsonAbstract:Previous work has shown that acetylcholinesterase (AChE), a member of the α/β-hydrolase superfamily, is stereoselectively inhibited by the four stereoisomers of Isomalathion. Recent kinetic and mass spectral data demonstrated that a difference in mechanism of inactivation exists for AChE treated with (1R)- versus (1S,3S)-stereoisomers. This study sought to determine whether other α/β-hydrolases are stereoselectively inhibited by Isomalathion and if the difference in mechanism of AChE inactivation between (1R)- and (1S,3S)-isomers is conserved for other α/β-hydrolases. Bimolecular rate constants of inhibition (ki) were measured for human and equine butyrylcholinesterase (HBChE and EBChE, respectively) and bovine cholesterol esterase (BCholE) with all four isomers. Isomalathion isomers inhibited these enzymes with the following order of potency: (1R,3R) > (1R,3S) > (1S,3R) ≥ (1S,3S). Ratios of ki values for the most potent to the least potent isomer were 10.5 (HBChE), 11.9 (EBChE), and 68.6 (BCholE). Rate ...
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inhibition of acetylcholinesterase by 1s 3s Isomalathion proceeds with loss of thiomethyl kinetic and mass spectral evidence for an unexpected primary leaving group
Chemical Research in Toxicology, 2000Co-Authors: Jonathan A Doorn, Charles M. Thompson, Todd T Talley, Michael Schall, Douglas A Gage, Rudy J. RichardsonAbstract:Previous work demonstrated kinetically that inhibition of mammalian acetylcholinesterase (AChE) by (1S)-Isomalathions may proceed by loss of thiomethyl instead of the expected diethyl thiosuccinate as the primary leaving group followed by one of four possible modes of rapid aging. This study sought to identify the adduct that renders AChE refractory toward reactivation after inhibition with the (1S, 3S)-stereoisomer. Electric eel acetylcholinesterase (EEAChE) was inhibited with the four stereoisomers of Isomalathion, and rate constants for spontaneous and oxime-mediated reactivation (k(3)) were measured. Oxime-mediated k(3) values were >25-fold higher for enzyme inhibited by (1R)- versus (1S)-stereoisomers with the greatest contrast between the (1R,3R)- and (1S,3S)-enantiomers. EEAChE inactivated by (1R,3R)-Isomalathion reactivated spontaneously and in the presence of pyridine-2-aldoxime methiodide (2-PAM) with k(3) values of 1.88 x 10(5) and 4.18 x 10(5) min(-)(1), respectively. In contrast, enzyme treated with the (1S,3S)-enantiomer had spontaneous and 2-PAM-mediated k(3) values of 0 and 6.05 x 10(3) min(-)(1), respectively. The kinetic data that were measured were consistent with those obtained for mammalian AChE used in previous studies. Identification of the adduct that renders EEAChE stable toward reactivation after inhibition with (1S,3S)-Isomalathion was accomplished using a peptide mass mapping approach with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A peak with a mass corresponding to the active site peptide containing the catalytic Ser with a covalently bound O-methyl phosphate adduct was found in the mass spectra of (1S, 3S)-treated EEAChE but not control samples. Identities of the modified active site peptide and adduct were confirmed by fragmentation in MALDI-TOF-MS post-source decay (PSD) analysis, and peaks corresponding to the loss of an adduct as phosphorous/phosphoric acid methyl ester were observed. The results demonstrate that inhibition of EEAChE by (1S,3S)-Isomalathion proceeds with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate as the secondary leaving group to yield an aged enzyme.
Charles M. Thompson - One of the best experts on this subject based on the ideXlab platform.
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inhibition of acetylcholinesterases by stereoisomeric organophosphorus compounds containing both thioester and p nitrophenyl leaving groups
Chemical Research in Toxicology, 2020Co-Authors: Todd T Talley, Rudy J. Richardson, Clifford E. Berkman, Chihkai Chao, Charles M. ThompsonAbstract:Studies with acetylcholinesterase (AChE) inhibited by organophosphorus (OP) compounds with two chiral centers can serve as models or surrogates for understanding the rate, orientation, and postinhibitory mechanisms by the nerve agent soman that possesses dual phosphorus and carbon chiral centers. In the current approach, stereoisomers of O-methyl, [S-(succinic acid, diethyl ester), O-(4-nitrophenyl) phosphorothiolate (MSNPs) were synthesized, and the inhibition, reactivation, and aging mechanisms were studied with electric eel AChE (eeAChE) and recombinant mouse brain AChE (rmAChE). The MSNP RPRC isomer was the strongest inhibitor of both eeAChE and rmAChE at 8- and 24-fold greater potency, respectively, than the weakest SPSC isomer. eeAChE inhibited by the RPRC- or RPSC-MSNP isomer underwent spontaneous reactivation ∼10- to 20-fold faster than the enzyme inhibited by SPRC- and SPSC-MSNP, and only 4% spontaneous reactivation was observed from the SPRC-eeAChE adduct. Using 2-pyridine aldoxime methiodide (2-PAM) or trimedoxime (TMB-4), eeAChE inhibited by RPRC- or SPRC-MSNP reactivated up to 90% and 3- to 4-fold faster than eeAChE inhibited by the RPSC- or SPSC-MSNP isomer. Spontaneous reactivation rates for rmAChE were 1.5- to 10-fold higher following inhibition by RPSC- and SPSC-MSNPs than inhibition by either RC isomer, a trend opposite to that found for eeAChE. Oxime reactivation of rmAChE following inhibition by RPRC- and SPRC-MSNPs was 2.5- to 5-fold faster than inhibition by RPSC- or SPSC-MSNPs. Due to structural similarities, MSNPs that phosphylate AChE with the loss of the p-nitrophenoxy (PNP) group form identical, nonreactivatable adducts to those formed from SP-Isomalathion; however, all the MSNP isomers inhibited AChE to form adducts that reactivated. Thus, MSNPs inactivate AChE via the ejection of either PNP or thiosuccinyl groups to form a combination of reactivatable and nonreactivatable adducts, and this differs from the mechanism of AChE inhibition by Isomalathion.
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stereoselective inactivation of torpedo californica acetylcholinesterase by Isomalathion inhibitory reactions with 1r and 1s isomers proceed by different mechanisms
Chemical Research in Toxicology, 2003Co-Authors: Jonathan A Doorn, Charles M. Thompson, Robert B Christner, Rudy J. RichardsonAbstract:The present study was undertaken to test the hypothesis that acetylcholinesterase (AChE) inhibition by Isomalathion stereoisomers proceeds with different primary leaving groups for (1R)- and (1S)-isomers. Consistent with results obtained with enzyme from other species, AChE from Torpedo californica (TcAChE) was stereoselectively inhibited by Isomalathion isomers with the (1R,3R)-isomer exhibiting greater potency than (1S,3S)-Isomalathion. TcAChE modified by (1R)-isomers readily reactivated in the presence of 2-pralidoxime methiodide (2-PAM), whereas enzyme inhibited by (1S)-Isomalathions was intractable toward reactivation. Computer-based molecular modeling showed that the ligand positioned as the primary leaving group was diethyl thiosuccinyl for (1R)-isomers and thiomethyl for (1S)-Isomalathions. Mass spectral analysis revealed that inhibition of TcAChE by (1R)-isomers resulted in an O,S-dimethyl phosphate adduct, as expected from expulsion of the diethyl thiosuccinyl ligand. In contrast, inactivation of the enzyme by (1S)-Isomalathions yielded an O-methyl phosphate adduct, consistent with initial loss of thiomethyl followed by displacement of the diethyl thiosuccinyl group. The findings demonstrate that the inhibitory reactions of TcAChE with (1R)- and (1S)-Isomalathions proceed by different mechanisms involving distinct primary leaving groups.
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identification of butyrylcholinesterase adducts after inhibition with Isomalathion using mass spectrometry difference in mechanism between 1r and 1s stereoisomers
Toxicology and Applied Pharmacology, 2001Co-Authors: Jonathan A Doorn, Charles M. Thompson, Todd T Talley, Michael Schall, Douglas A Gage, Rudy J. RichardsonAbstract:Previous kinetic studies found that butyrylcholinesterase (BChE) inhibited by (1R)-Isomalathions readily reactivated, while enzyme inactivated by (1S)-isomers did not. This study tested the hypothesis that (1R)- and (1S)-isomers inhibit BChE by different mechanisms, yielding distinct adducts identifiable by peptide mass mapping with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS). Equine BChE (EBChE) was inhibited to <10% of control activity with each isomer of Isomalathion and the reference compound isoparathion methyl. Control and treated enzyme was digested with trypsin, and peptides were fractionated with HPLC. Separated and unseparated peptides were analyzed with MALDI–TOF–MS. Identity of an organophosphorus peptide adduct was confirmed by fragmentation using postsource decay analysis. EBChE inhibited by (1R)-Isomalathions or (S)-isoparathion methyl readily reactivated after oxime treatment with 30–40% activity recovered. Enzyme inactivated by (1S)-Isomalathions or (R)-isoparathion methyl recovered <2% and <5% activity, respectively, after oxime treatment. MALDI–TOF–MS analysis revealed that inhibition of EBChE by (1R)-Isomalathions and (R)- or (S)-isoparathion methyl yielded O,S-dimethyl phosphate adducts. Enzyme inactivated by (1S)-Isomalathions produced only O-methyl phosphate adduct. EBChE modified by (1R)-Isomalathions or either enantiomer of isoparathion methyl yielded an O-methyl phosphate adduct as well. The results indicate that EBChE inhibition by (1R)-Isomalathions proceeds with loss of diethyl thiosuccinate, but inactivation by (1S)-isomers occurs with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate to yield an aged enzyme. Furthermore, the data suggest that aging of the O,S-dimethyl phosphate adduct occurs via an SN2 process with loss of thiomethyl.
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probing the active sites of butyrylcholinesterase and cholesterol esterase with Isomalathion conserved stereoselective inactivation of serine hydrolases structurally related to acetylcholinesterase
Chemical Research in Toxicology, 2001Co-Authors: Jonathan A Doorn, Todd T Talley, Charles M. Thompson, Rudy J. RichardsonAbstract:Previous work has shown that acetylcholinesterase (AChE), a member of the α/β-hydrolase superfamily, is stereoselectively inhibited by the four stereoisomers of Isomalathion. Recent kinetic and mass spectral data demonstrated that a difference in mechanism of inactivation exists for AChE treated with (1R)- versus (1S,3S)-stereoisomers. This study sought to determine whether other α/β-hydrolases are stereoselectively inhibited by Isomalathion and if the difference in mechanism of AChE inactivation between (1R)- and (1S,3S)-isomers is conserved for other α/β-hydrolases. Bimolecular rate constants of inhibition (ki) were measured for human and equine butyrylcholinesterase (HBChE and EBChE, respectively) and bovine cholesterol esterase (BCholE) with all four isomers. Isomalathion isomers inhibited these enzymes with the following order of potency: (1R,3R) > (1R,3S) > (1S,3R) ≥ (1S,3S). Ratios of ki values for the most potent to the least potent isomer were 10.5 (HBChE), 11.9 (EBChE), and 68.6 (BCholE). Rate ...
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inhibition of acetylcholinesterase by 1s 3s Isomalathion proceeds with loss of thiomethyl kinetic and mass spectral evidence for an unexpected primary leaving group
Chemical Research in Toxicology, 2000Co-Authors: Jonathan A Doorn, Charles M. Thompson, Todd T Talley, Michael Schall, Douglas A Gage, Rudy J. RichardsonAbstract:Previous work demonstrated kinetically that inhibition of mammalian acetylcholinesterase (AChE) by (1S)-Isomalathions may proceed by loss of thiomethyl instead of the expected diethyl thiosuccinate as the primary leaving group followed by one of four possible modes of rapid aging. This study sought to identify the adduct that renders AChE refractory toward reactivation after inhibition with the (1S, 3S)-stereoisomer. Electric eel acetylcholinesterase (EEAChE) was inhibited with the four stereoisomers of Isomalathion, and rate constants for spontaneous and oxime-mediated reactivation (k(3)) were measured. Oxime-mediated k(3) values were >25-fold higher for enzyme inhibited by (1R)- versus (1S)-stereoisomers with the greatest contrast between the (1R,3R)- and (1S,3S)-enantiomers. EEAChE inactivated by (1R,3R)-Isomalathion reactivated spontaneously and in the presence of pyridine-2-aldoxime methiodide (2-PAM) with k(3) values of 1.88 x 10(5) and 4.18 x 10(5) min(-)(1), respectively. In contrast, enzyme treated with the (1S,3S)-enantiomer had spontaneous and 2-PAM-mediated k(3) values of 0 and 6.05 x 10(3) min(-)(1), respectively. The kinetic data that were measured were consistent with those obtained for mammalian AChE used in previous studies. Identification of the adduct that renders EEAChE stable toward reactivation after inhibition with (1S,3S)-Isomalathion was accomplished using a peptide mass mapping approach with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A peak with a mass corresponding to the active site peptide containing the catalytic Ser with a covalently bound O-methyl phosphate adduct was found in the mass spectra of (1S, 3S)-treated EEAChE but not control samples. Identities of the modified active site peptide and adduct were confirmed by fragmentation in MALDI-TOF-MS post-source decay (PSD) analysis, and peaks corresponding to the loss of an adduct as phosphorous/phosphoric acid methyl ester were observed. The results demonstrate that inhibition of EEAChE by (1S,3S)-Isomalathion proceeds with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate as the secondary leaving group to yield an aged enzyme.
Clifford E. Berkman - One of the best experts on this subject based on the ideXlab platform.
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inhibition of acetylcholinesterases by stereoisomeric organophosphorus compounds containing both thioester and p nitrophenyl leaving groups
Chemical Research in Toxicology, 2020Co-Authors: Todd T Talley, Rudy J. Richardson, Clifford E. Berkman, Chihkai Chao, Charles M. ThompsonAbstract:Studies with acetylcholinesterase (AChE) inhibited by organophosphorus (OP) compounds with two chiral centers can serve as models or surrogates for understanding the rate, orientation, and postinhibitory mechanisms by the nerve agent soman that possesses dual phosphorus and carbon chiral centers. In the current approach, stereoisomers of O-methyl, [S-(succinic acid, diethyl ester), O-(4-nitrophenyl) phosphorothiolate (MSNPs) were synthesized, and the inhibition, reactivation, and aging mechanisms were studied with electric eel AChE (eeAChE) and recombinant mouse brain AChE (rmAChE). The MSNP RPRC isomer was the strongest inhibitor of both eeAChE and rmAChE at 8- and 24-fold greater potency, respectively, than the weakest SPSC isomer. eeAChE inhibited by the RPRC- or RPSC-MSNP isomer underwent spontaneous reactivation ∼10- to 20-fold faster than the enzyme inhibited by SPRC- and SPSC-MSNP, and only 4% spontaneous reactivation was observed from the SPRC-eeAChE adduct. Using 2-pyridine aldoxime methiodide (2-PAM) or trimedoxime (TMB-4), eeAChE inhibited by RPRC- or SPRC-MSNP reactivated up to 90% and 3- to 4-fold faster than eeAChE inhibited by the RPSC- or SPSC-MSNP isomer. Spontaneous reactivation rates for rmAChE were 1.5- to 10-fold higher following inhibition by RPSC- and SPSC-MSNPs than inhibition by either RC isomer, a trend opposite to that found for eeAChE. Oxime reactivation of rmAChE following inhibition by RPRC- and SPRC-MSNPs was 2.5- to 5-fold faster than inhibition by RPSC- or SPSC-MSNPs. Due to structural similarities, MSNPs that phosphylate AChE with the loss of the p-nitrophenoxy (PNP) group form identical, nonreactivatable adducts to those formed from SP-Isomalathion; however, all the MSNP isomers inhibited AChE to form adducts that reactivated. Thus, MSNPs inactivate AChE via the ejection of either PNP or thiosuccinyl groups to form a combination of reactivatable and nonreactivatable adducts, and this differs from the mechanism of AChE inhibition by Isomalathion.
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kinetic evidence for different mechanisms of acetylcholinesterase inhibition by 1r and 1s stereoisomers of Isomalathion
Toxicology and Applied Pharmacology, 1999Co-Authors: Suree Jianmongkol, Clifford E. Berkman, Charles M. Thompson, Brian R Marable, Todd T Talley, Rudy J. RichardsonAbstract:Abstract Inhibition of acetylcholinesterase (AChE) by Isomalathion has been assumed to proceed by expulsion of diethyl thiosuccinyl to produce O,S -dimethyl phosphorylated AChE. If this assumption is correct, AChE inhibited by (1 R )- or (1 S )-Isomalathions should reactivate at the same rate as AChE inhibited by configurationally equivalent ( S )- or ( R )-isoparathion methyl, respectively, which are expected to inhibit AChE by loss of 4-nitrophenoxyl to yield O,S -dimethyl phosphorylated AChEs. Previous work has shown that rat brain AChE inhibited by (1 R )-Isomalathions reactivates at the same rate as the enzyme inhibited by ( S )-isoparathion methyl. However, although rat brain AChE inhibited by ( R )-isoparathion methyl reactivates at a measurable rate, the enzyme inhibited by (1 S )-Isomalathions is intractable to reactivation. This surprising finding suggests the hypothesis that (1 R )- and (1 S )-stereoisomers of Isomalathion inhibit AChE by different mechanisms, yielding enzymatic species distinguishable by their postinhibitory kinetics. The present study was carried out to test this hypothesis by comparing kinetic constants of reactivation ( k +3 ) and aging ( k +4 ) of hen brain AChE and bovine erythrocyte AChE inhibited by the four stereoisomers of Isomalathion and the two stereoisomers of isoparathion methyl. Both AChEs inhibited by either (1 R ,3 R )- or (1 R ,3 S )-Isomalathion had comparable corresponding k +3 values (spontaneous and oxime-mediated) to those of AChEs inhibited with ( S )-isoparathion methyl. However, spontaneous and oxime-mediated k +3 values comparable to those of ( R )-isoparathion methyl could not be obtained for AChEs inhibited by (1 S ,3 R )- and (1 S ,3 S )-Isomalathion. Comparison of k +4 values for hen brain AChE inhibited by each stereoisomer of Isomalathion and isoparathion methyl corroborated that only the (1 S )-Isomalathions failed to produce the expected O,S -dimethyl phosphoryl-conjugated enzymes. The results for (1 R )-Isomalathions suggest that the mechanism of inhibition of AChE by these isomers is the expected one involving diethyl thiosuccinyl as the primary leaving group. In contrast, the results for (1 S )-Isomalathions are consistent with an alternative mechanism of inhibition by these isomers implicating loss of thiomethyl as the primary leaving group.
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Relative potencies of the four stereoisomers of Isomalathion for inhibition of hen brain acetylcholinesterase and neurotoxic esterase in vitro
Toxicology and Applied Pharmacology, 1996Co-Authors: Suree Jianmongkol, Clifford E. Berkman, Charles M. Thompson, Rudy J. RichardsonAbstract:The cholinergic toxicity of malathion is exacerbated by its isomerization product, Isomalathion, which inhibits detoxifying carboxylesterases as well as target acetylcholinesterase (AChE). Previous work has shown that the four stereoisomers of Isomalathion, (1R, 3R), (1R, 3S), (1S, 3R), and (1S, 3S), differ in their inhibitory potencies against either rat brain or electric eel AChE. The present study examined the relative inhibitory potencies of these stereoisomers and the totally racemic mixture (1RS, 3RS) against hen brain AChE and neurotoxic esterase (NTE) to provide new data on stereoselective inhibition of neurotoxicologically significant esterases and to assess the potential of these compounds to cause organophosphorus (OP) compound-induced delayed neurotoxicity (OPIDN). The order of potencies against hen brain AChE was (1R, 3R) > (1R, 3S) > (1RS, 3RS) > (1S, 3R) > (1S, 3S), with a 15-fold difference between the strongest (ki = 388 mM-1 min-1; 20 min I50 = 89.3 nM) and weakest (ki = 25.6 mM-1 min-1; 20 min I50 = 1354 nM) inhibitors. Both asymmetric centers contributed substantially and interdependently to inhibitory potency, but the effect of changing the configuration at phosphorus alone was greater than changing the configuration at carbon alone. None of the Isomalathions was an effective inhibitor of hen brain NTE (extrapolated 20 min I50 values were 1.2 to 29 mM), yielding NTE/ AChE I50 ratios (neuropathy target ratios, NTRs) of 1.5 x 10(3) to 1.5 x 10(5). NTRs of this magnitude indicate that none of the Isomalathions should initiate OPIDN, even after doses greatly exceeding the LD50. Therefore, reports of OPIDN or other neuropathic sequelae associated with malathion exposures in humans cannot be explained on the basis of NTE inhibition by contaminating Isomalathions.
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Synthesis, absolute configuration, and analysis of malathion, malaoxon, and Isomalathion enantiomers
Chemical research in toxicology, 1993Co-Authors: Clifford E. Berkman, Charles M. Thompson, Scott R. PerrinAbstract:Syntheses of the enantiomers of malathion, malaoxon, and Isomalathion are reported herein. Malathion enantiomers were prepared from (R)- or (S)-malic acid in three steps. Enantiomers of malathion were converted to the corresponding enantiomers of malaoxon in 52% yield by oxidation with monoperoxyphthalic acid, magnesium salt. The four Isomalathion stereoisomers were prepared via two independent pathways using strychnine to resolve the asymmetric phosphorus moiety. The absolute configurations of the four stereoisomers of Isomalathion were determined by X-ray crystallographic analysis of an alkaloid salt precursor. A high-performance liquid chromatography technique was developed to resolve the four stereoisomers of Isomalathion, and to determine their stereoisomeric ratios.
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Interaction of acetylcholinesterase with the enantiomers of malaoxon and Isomalathion
Chemical research in toxicology, 1993Co-Authors: Clifford E. Berkman, Debra A. Quinn, Charles M. ThompsonAbstract:The biomolecular reaction constants (ki), dissociation constants (Kd), and phosphorylation constants (kp) were determined for the enantiomers of malaoxon against rat brain acetylcholinesterase, and for the stereoisomers of Isomalathion against rat brain acetylcholinesterase and electric eel acetylcholinesterase. (R)-Malaoxon was an 8.6-fold more potent anti-cholinesterase than (S)-malaoxon. Isomalathion stereoisomers with the R configuration at carbon were 3-13-fold stronger inhibitors than those with the S configuration. The Isomalathion stereoisomers with the R configuration at phosphorus were 4.3-8.8-fold stronger inhibitors of rat brain acetylcholinesterase, yet 3.4-5.8-fold weaker inhibitors of electric eel acetylcholinesterase, than the Isomalathion stereoisomers with the S configuration at phosphorus. The rat brain acetylcholinesterase spontaneous (k0 = approximately 13.0 x 10(-3) min-1) and oxime-mediated (koxime) = 51.0 x 10(-3) min-1) reactivation rate constants following inhibition by Isomalathion stereoisomers with the R configuration at phosphorus were comparable to spontaneous (11.3 x 10(-3) min-1) and oxime-mediated (50.2 x 10(-3) min-1) reactivation rates obtained for (S)-isoparathion methyl. These data support a common phosphorylation mechanism, namely, the displacement of the thiosuccinyl moiety from Isomalathion stereoisomers with the R configuration at phosphorus, and displacement of the p-nitrophenoxy ligand from (S)-isoparathion methyl to form the same O,S-dimethyl phosphorothiolated enzyme. Rat brain acetylcholinesterase inhibited by the Isomalathion stereoisomers with the S configuration at phosphorus were refractory to reactivation, suggesting an alternate mechanism of inhibition, i.e., the loss of the methylthio ligand. Several mechanisms are proposed to account for the subsequent nonreactivation.(ABSTRACT TRUNCATED AT 250 WORDS)
Jonathan A Doorn - One of the best experts on this subject based on the ideXlab platform.
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stereoselective inactivation of torpedo californica acetylcholinesterase by Isomalathion inhibitory reactions with 1r and 1s isomers proceed by different mechanisms
Chemical Research in Toxicology, 2003Co-Authors: Jonathan A Doorn, Charles M. Thompson, Robert B Christner, Rudy J. RichardsonAbstract:The present study was undertaken to test the hypothesis that acetylcholinesterase (AChE) inhibition by Isomalathion stereoisomers proceeds with different primary leaving groups for (1R)- and (1S)-isomers. Consistent with results obtained with enzyme from other species, AChE from Torpedo californica (TcAChE) was stereoselectively inhibited by Isomalathion isomers with the (1R,3R)-isomer exhibiting greater potency than (1S,3S)-Isomalathion. TcAChE modified by (1R)-isomers readily reactivated in the presence of 2-pralidoxime methiodide (2-PAM), whereas enzyme inhibited by (1S)-Isomalathions was intractable toward reactivation. Computer-based molecular modeling showed that the ligand positioned as the primary leaving group was diethyl thiosuccinyl for (1R)-isomers and thiomethyl for (1S)-Isomalathions. Mass spectral analysis revealed that inhibition of TcAChE by (1R)-isomers resulted in an O,S-dimethyl phosphate adduct, as expected from expulsion of the diethyl thiosuccinyl ligand. In contrast, inactivation of the enzyme by (1S)-Isomalathions yielded an O-methyl phosphate adduct, consistent with initial loss of thiomethyl followed by displacement of the diethyl thiosuccinyl group. The findings demonstrate that the inhibitory reactions of TcAChE with (1R)- and (1S)-Isomalathions proceed by different mechanisms involving distinct primary leaving groups.
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identification of butyrylcholinesterase adducts after inhibition with Isomalathion using mass spectrometry difference in mechanism between 1r and 1s stereoisomers
Toxicology and Applied Pharmacology, 2001Co-Authors: Jonathan A Doorn, Charles M. Thompson, Todd T Talley, Michael Schall, Douglas A Gage, Rudy J. RichardsonAbstract:Previous kinetic studies found that butyrylcholinesterase (BChE) inhibited by (1R)-Isomalathions readily reactivated, while enzyme inactivated by (1S)-isomers did not. This study tested the hypothesis that (1R)- and (1S)-isomers inhibit BChE by different mechanisms, yielding distinct adducts identifiable by peptide mass mapping with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS). Equine BChE (EBChE) was inhibited to <10% of control activity with each isomer of Isomalathion and the reference compound isoparathion methyl. Control and treated enzyme was digested with trypsin, and peptides were fractionated with HPLC. Separated and unseparated peptides were analyzed with MALDI–TOF–MS. Identity of an organophosphorus peptide adduct was confirmed by fragmentation using postsource decay analysis. EBChE inhibited by (1R)-Isomalathions or (S)-isoparathion methyl readily reactivated after oxime treatment with 30–40% activity recovered. Enzyme inactivated by (1S)-Isomalathions or (R)-isoparathion methyl recovered <2% and <5% activity, respectively, after oxime treatment. MALDI–TOF–MS analysis revealed that inhibition of EBChE by (1R)-Isomalathions and (R)- or (S)-isoparathion methyl yielded O,S-dimethyl phosphate adducts. Enzyme inactivated by (1S)-Isomalathions produced only O-methyl phosphate adduct. EBChE modified by (1R)-Isomalathions or either enantiomer of isoparathion methyl yielded an O-methyl phosphate adduct as well. The results indicate that EBChE inhibition by (1R)-Isomalathions proceeds with loss of diethyl thiosuccinate, but inactivation by (1S)-isomers occurs with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate to yield an aged enzyme. Furthermore, the data suggest that aging of the O,S-dimethyl phosphate adduct occurs via an SN2 process with loss of thiomethyl.
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probing the active sites of butyrylcholinesterase and cholesterol esterase with Isomalathion conserved stereoselective inactivation of serine hydrolases structurally related to acetylcholinesterase
Chemical Research in Toxicology, 2001Co-Authors: Jonathan A Doorn, Todd T Talley, Charles M. Thompson, Rudy J. RichardsonAbstract:Previous work has shown that acetylcholinesterase (AChE), a member of the α/β-hydrolase superfamily, is stereoselectively inhibited by the four stereoisomers of Isomalathion. Recent kinetic and mass spectral data demonstrated that a difference in mechanism of inactivation exists for AChE treated with (1R)- versus (1S,3S)-stereoisomers. This study sought to determine whether other α/β-hydrolases are stereoselectively inhibited by Isomalathion and if the difference in mechanism of AChE inactivation between (1R)- and (1S,3S)-isomers is conserved for other α/β-hydrolases. Bimolecular rate constants of inhibition (ki) were measured for human and equine butyrylcholinesterase (HBChE and EBChE, respectively) and bovine cholesterol esterase (BCholE) with all four isomers. Isomalathion isomers inhibited these enzymes with the following order of potency: (1R,3R) > (1R,3S) > (1S,3R) ≥ (1S,3S). Ratios of ki values for the most potent to the least potent isomer were 10.5 (HBChE), 11.9 (EBChE), and 68.6 (BCholE). Rate ...
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inhibition of acetylcholinesterase by 1s 3s Isomalathion proceeds with loss of thiomethyl kinetic and mass spectral evidence for an unexpected primary leaving group
Chemical Research in Toxicology, 2000Co-Authors: Jonathan A Doorn, Charles M. Thompson, Todd T Talley, Michael Schall, Douglas A Gage, Rudy J. RichardsonAbstract:Previous work demonstrated kinetically that inhibition of mammalian acetylcholinesterase (AChE) by (1S)-Isomalathions may proceed by loss of thiomethyl instead of the expected diethyl thiosuccinate as the primary leaving group followed by one of four possible modes of rapid aging. This study sought to identify the adduct that renders AChE refractory toward reactivation after inhibition with the (1S, 3S)-stereoisomer. Electric eel acetylcholinesterase (EEAChE) was inhibited with the four stereoisomers of Isomalathion, and rate constants for spontaneous and oxime-mediated reactivation (k(3)) were measured. Oxime-mediated k(3) values were >25-fold higher for enzyme inhibited by (1R)- versus (1S)-stereoisomers with the greatest contrast between the (1R,3R)- and (1S,3S)-enantiomers. EEAChE inactivated by (1R,3R)-Isomalathion reactivated spontaneously and in the presence of pyridine-2-aldoxime methiodide (2-PAM) with k(3) values of 1.88 x 10(5) and 4.18 x 10(5) min(-)(1), respectively. In contrast, enzyme treated with the (1S,3S)-enantiomer had spontaneous and 2-PAM-mediated k(3) values of 0 and 6.05 x 10(3) min(-)(1), respectively. The kinetic data that were measured were consistent with those obtained for mammalian AChE used in previous studies. Identification of the adduct that renders EEAChE stable toward reactivation after inhibition with (1S,3S)-Isomalathion was accomplished using a peptide mass mapping approach with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A peak with a mass corresponding to the active site peptide containing the catalytic Ser with a covalently bound O-methyl phosphate adduct was found in the mass spectra of (1S, 3S)-treated EEAChE but not control samples. Identities of the modified active site peptide and adduct were confirmed by fragmentation in MALDI-TOF-MS post-source decay (PSD) analysis, and peaks corresponding to the loss of an adduct as phosphorous/phosphoric acid methyl ester were observed. The results demonstrate that inhibition of EEAChE by (1S,3S)-Isomalathion proceeds with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate as the secondary leaving group to yield an aged enzyme.
Todd T Talley - One of the best experts on this subject based on the ideXlab platform.
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inhibition of acetylcholinesterases by stereoisomeric organophosphorus compounds containing both thioester and p nitrophenyl leaving groups
Chemical Research in Toxicology, 2020Co-Authors: Todd T Talley, Rudy J. Richardson, Clifford E. Berkman, Chihkai Chao, Charles M. ThompsonAbstract:Studies with acetylcholinesterase (AChE) inhibited by organophosphorus (OP) compounds with two chiral centers can serve as models or surrogates for understanding the rate, orientation, and postinhibitory mechanisms by the nerve agent soman that possesses dual phosphorus and carbon chiral centers. In the current approach, stereoisomers of O-methyl, [S-(succinic acid, diethyl ester), O-(4-nitrophenyl) phosphorothiolate (MSNPs) were synthesized, and the inhibition, reactivation, and aging mechanisms were studied with electric eel AChE (eeAChE) and recombinant mouse brain AChE (rmAChE). The MSNP RPRC isomer was the strongest inhibitor of both eeAChE and rmAChE at 8- and 24-fold greater potency, respectively, than the weakest SPSC isomer. eeAChE inhibited by the RPRC- or RPSC-MSNP isomer underwent spontaneous reactivation ∼10- to 20-fold faster than the enzyme inhibited by SPRC- and SPSC-MSNP, and only 4% spontaneous reactivation was observed from the SPRC-eeAChE adduct. Using 2-pyridine aldoxime methiodide (2-PAM) or trimedoxime (TMB-4), eeAChE inhibited by RPRC- or SPRC-MSNP reactivated up to 90% and 3- to 4-fold faster than eeAChE inhibited by the RPSC- or SPSC-MSNP isomer. Spontaneous reactivation rates for rmAChE were 1.5- to 10-fold higher following inhibition by RPSC- and SPSC-MSNPs than inhibition by either RC isomer, a trend opposite to that found for eeAChE. Oxime reactivation of rmAChE following inhibition by RPRC- and SPRC-MSNPs was 2.5- to 5-fold faster than inhibition by RPSC- or SPSC-MSNPs. Due to structural similarities, MSNPs that phosphylate AChE with the loss of the p-nitrophenoxy (PNP) group form identical, nonreactivatable adducts to those formed from SP-Isomalathion; however, all the MSNP isomers inhibited AChE to form adducts that reactivated. Thus, MSNPs inactivate AChE via the ejection of either PNP or thiosuccinyl groups to form a combination of reactivatable and nonreactivatable adducts, and this differs from the mechanism of AChE inhibition by Isomalathion.
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identification of butyrylcholinesterase adducts after inhibition with Isomalathion using mass spectrometry difference in mechanism between 1r and 1s stereoisomers
Toxicology and Applied Pharmacology, 2001Co-Authors: Jonathan A Doorn, Charles M. Thompson, Todd T Talley, Michael Schall, Douglas A Gage, Rudy J. RichardsonAbstract:Previous kinetic studies found that butyrylcholinesterase (BChE) inhibited by (1R)-Isomalathions readily reactivated, while enzyme inactivated by (1S)-isomers did not. This study tested the hypothesis that (1R)- and (1S)-isomers inhibit BChE by different mechanisms, yielding distinct adducts identifiable by peptide mass mapping with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS). Equine BChE (EBChE) was inhibited to <10% of control activity with each isomer of Isomalathion and the reference compound isoparathion methyl. Control and treated enzyme was digested with trypsin, and peptides were fractionated with HPLC. Separated and unseparated peptides were analyzed with MALDI–TOF–MS. Identity of an organophosphorus peptide adduct was confirmed by fragmentation using postsource decay analysis. EBChE inhibited by (1R)-Isomalathions or (S)-isoparathion methyl readily reactivated after oxime treatment with 30–40% activity recovered. Enzyme inactivated by (1S)-Isomalathions or (R)-isoparathion methyl recovered <2% and <5% activity, respectively, after oxime treatment. MALDI–TOF–MS analysis revealed that inhibition of EBChE by (1R)-Isomalathions and (R)- or (S)-isoparathion methyl yielded O,S-dimethyl phosphate adducts. Enzyme inactivated by (1S)-Isomalathions produced only O-methyl phosphate adduct. EBChE modified by (1R)-Isomalathions or either enantiomer of isoparathion methyl yielded an O-methyl phosphate adduct as well. The results indicate that EBChE inhibition by (1R)-Isomalathions proceeds with loss of diethyl thiosuccinate, but inactivation by (1S)-isomers occurs with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate to yield an aged enzyme. Furthermore, the data suggest that aging of the O,S-dimethyl phosphate adduct occurs via an SN2 process with loss of thiomethyl.
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probing the active sites of butyrylcholinesterase and cholesterol esterase with Isomalathion conserved stereoselective inactivation of serine hydrolases structurally related to acetylcholinesterase
Chemical Research in Toxicology, 2001Co-Authors: Jonathan A Doorn, Todd T Talley, Charles M. Thompson, Rudy J. RichardsonAbstract:Previous work has shown that acetylcholinesterase (AChE), a member of the α/β-hydrolase superfamily, is stereoselectively inhibited by the four stereoisomers of Isomalathion. Recent kinetic and mass spectral data demonstrated that a difference in mechanism of inactivation exists for AChE treated with (1R)- versus (1S,3S)-stereoisomers. This study sought to determine whether other α/β-hydrolases are stereoselectively inhibited by Isomalathion and if the difference in mechanism of AChE inactivation between (1R)- and (1S,3S)-isomers is conserved for other α/β-hydrolases. Bimolecular rate constants of inhibition (ki) were measured for human and equine butyrylcholinesterase (HBChE and EBChE, respectively) and bovine cholesterol esterase (BCholE) with all four isomers. Isomalathion isomers inhibited these enzymes with the following order of potency: (1R,3R) > (1R,3S) > (1S,3R) ≥ (1S,3S). Ratios of ki values for the most potent to the least potent isomer were 10.5 (HBChE), 11.9 (EBChE), and 68.6 (BCholE). Rate ...
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inhibition of acetylcholinesterase by 1s 3s Isomalathion proceeds with loss of thiomethyl kinetic and mass spectral evidence for an unexpected primary leaving group
Chemical Research in Toxicology, 2000Co-Authors: Jonathan A Doorn, Charles M. Thompson, Todd T Talley, Michael Schall, Douglas A Gage, Rudy J. RichardsonAbstract:Previous work demonstrated kinetically that inhibition of mammalian acetylcholinesterase (AChE) by (1S)-Isomalathions may proceed by loss of thiomethyl instead of the expected diethyl thiosuccinate as the primary leaving group followed by one of four possible modes of rapid aging. This study sought to identify the adduct that renders AChE refractory toward reactivation after inhibition with the (1S, 3S)-stereoisomer. Electric eel acetylcholinesterase (EEAChE) was inhibited with the four stereoisomers of Isomalathion, and rate constants for spontaneous and oxime-mediated reactivation (k(3)) were measured. Oxime-mediated k(3) values were >25-fold higher for enzyme inhibited by (1R)- versus (1S)-stereoisomers with the greatest contrast between the (1R,3R)- and (1S,3S)-enantiomers. EEAChE inactivated by (1R,3R)-Isomalathion reactivated spontaneously and in the presence of pyridine-2-aldoxime methiodide (2-PAM) with k(3) values of 1.88 x 10(5) and 4.18 x 10(5) min(-)(1), respectively. In contrast, enzyme treated with the (1S,3S)-enantiomer had spontaneous and 2-PAM-mediated k(3) values of 0 and 6.05 x 10(3) min(-)(1), respectively. The kinetic data that were measured were consistent with those obtained for mammalian AChE used in previous studies. Identification of the adduct that renders EEAChE stable toward reactivation after inhibition with (1S,3S)-Isomalathion was accomplished using a peptide mass mapping approach with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A peak with a mass corresponding to the active site peptide containing the catalytic Ser with a covalently bound O-methyl phosphate adduct was found in the mass spectra of (1S, 3S)-treated EEAChE but not control samples. Identities of the modified active site peptide and adduct were confirmed by fragmentation in MALDI-TOF-MS post-source decay (PSD) analysis, and peaks corresponding to the loss of an adduct as phosphorous/phosphoric acid methyl ester were observed. The results demonstrate that inhibition of EEAChE by (1S,3S)-Isomalathion proceeds with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate as the secondary leaving group to yield an aged enzyme.
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kinetic evidence for different mechanisms of acetylcholinesterase inhibition by 1r and 1s stereoisomers of Isomalathion
Toxicology and Applied Pharmacology, 1999Co-Authors: Suree Jianmongkol, Clifford E. Berkman, Charles M. Thompson, Brian R Marable, Todd T Talley, Rudy J. RichardsonAbstract:Abstract Inhibition of acetylcholinesterase (AChE) by Isomalathion has been assumed to proceed by expulsion of diethyl thiosuccinyl to produce O,S -dimethyl phosphorylated AChE. If this assumption is correct, AChE inhibited by (1 R )- or (1 S )-Isomalathions should reactivate at the same rate as AChE inhibited by configurationally equivalent ( S )- or ( R )-isoparathion methyl, respectively, which are expected to inhibit AChE by loss of 4-nitrophenoxyl to yield O,S -dimethyl phosphorylated AChEs. Previous work has shown that rat brain AChE inhibited by (1 R )-Isomalathions reactivates at the same rate as the enzyme inhibited by ( S )-isoparathion methyl. However, although rat brain AChE inhibited by ( R )-isoparathion methyl reactivates at a measurable rate, the enzyme inhibited by (1 S )-Isomalathions is intractable to reactivation. This surprising finding suggests the hypothesis that (1 R )- and (1 S )-stereoisomers of Isomalathion inhibit AChE by different mechanisms, yielding enzymatic species distinguishable by their postinhibitory kinetics. The present study was carried out to test this hypothesis by comparing kinetic constants of reactivation ( k +3 ) and aging ( k +4 ) of hen brain AChE and bovine erythrocyte AChE inhibited by the four stereoisomers of Isomalathion and the two stereoisomers of isoparathion methyl. Both AChEs inhibited by either (1 R ,3 R )- or (1 R ,3 S )-Isomalathion had comparable corresponding k +3 values (spontaneous and oxime-mediated) to those of AChEs inhibited with ( S )-isoparathion methyl. However, spontaneous and oxime-mediated k +3 values comparable to those of ( R )-isoparathion methyl could not be obtained for AChEs inhibited by (1 S ,3 R )- and (1 S ,3 S )-Isomalathion. Comparison of k +4 values for hen brain AChE inhibited by each stereoisomer of Isomalathion and isoparathion methyl corroborated that only the (1 S )-Isomalathions failed to produce the expected O,S -dimethyl phosphoryl-conjugated enzymes. The results for (1 R )-Isomalathions suggest that the mechanism of inhibition of AChE by these isomers is the expected one involving diethyl thiosuccinyl as the primary leaving group. In contrast, the results for (1 S )-Isomalathions are consistent with an alternative mechanism of inhibition by these isomers implicating loss of thiomethyl as the primary leaving group.
