The Role of Glutamate-199 in the Aging of Cholinesterase

Saxena, Ashima; Doctor, B.P.; Maxwell, Donald M.; Lenz, David E.; Radić, Zoran; Taylor, Palmer

doi:10.1006/bbrc.1993.2481

Cited by 54 publications

(61 citation statements)

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“…Thus, these results are consistent with the structure and interaction energy calculations, which suggested that Glu202 stabilizes the O-isopropyl moiety of the sarin adduct in a conformation that enables nucleophilic attack on the phosphorus atom by HI-6. Glu202 has previously been reported as a key residue of AChE with an important role in catalysis (21), phosphylation (22), and aging (23). Here, we also observed, in addition to the interaction with the O-isopropyl moiety, an interaction between Glu202 and the methylene of Ser203 with an interaction energy of ∼7 kcal/mol (Table S4).…”

Section: Reactivation Kinetics Measurements Show the Importance Of Glsupporting

confidence: 72%

Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6

Allgardsson

Berg

Akfur

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Organophosphorus nerve agents interfere with cholinergic signaling by covalently binding to the active site of the enzyme acetylcholinesterase (AChE). This inhibition causes an accumulation of the neurotransmitter acetylcholine, potentially leading to overstimulation of the nervous system and death. Current treatments include the use of antidotes that promote the release of functional AChE by an unknown reactivation mechanism. We have used diffusion trap cryocrystallography and density functional theory (DFT) calculations to determine and analyze prereaction conformers of the nerve agent antidote HI-6 in complex with Mus musculus AChE covalently inhibited by the nerve agent sarin. These analyses reveal previously unknown conformations of the system and suggest that the cleavage of the covalent enzyme-sarin bond is preceded by a conformational change in the sarin adduct itself. Together with data from the reactivation kinetics, this alternate conformation suggests a key interaction between Glu202 and the O-isopropyl moiety of sarin. Moreover, solvent kinetic isotope effect experiments using deuterium oxide reveal that the reactivation mechanism features an isotope-sensitive step. These findings provide insights into the reactivation mechanism and provide a starting point for the development of improved antidotes. The work also illustrates how DFT calculations can guide the interpretation, analysis, and validation of crystallographic data for challenging reactive systems with complex conformational dynamics.acetylcholinesterase | density functional theory | crystallography | nerve agent | reactivation

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Section: Reactivation Kinetics Measurements Show the Importance Of Glsupporting

confidence: 72%

Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6

Allgardsson

Berg

Akfur

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Other important residues involved in the aging process of phosphylated cholinesterases have been identified : Glu-199 in somanphosphonylated Torpedo AChE [49] and Glu-202 in human AChE [50] (both are equivalent to Glu-197 in human BuChE) and Trp-84 in soman-phosphonylated human AChE [51] (equivalent to Trp-82 in human BuChE). These residues are in close contact with the alkyl chain (the pinacolyl chain in soman) which is removed from the phosphyl moiety during aging and have been found to be involved in stabilization of the developing carbonium intermediate.…”

Section: Agingmentioning

confidence: 99%

Importance of aspartate-70 in organophosphate inhibition, oxime re-activation and aging of human butyrylcholinesterase

Masson

Froment

Bartels

et al. 1997

Biochemical Journal

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Asp-70 is the defining amino acid in the peripheral anionic site of human butyrylcholinesterase (BuChE), whereas acetylcholinesterase has several additional amino acids, the most important one being Trp-277 (Trp-279 in TorpedoAChE). We studied mutants D70G, D70K and A277W to evaluate the role of Asp-70 and Trp-277 in reactions with organophosphates. We found that Asp-70 was important for binding positively charged echothiophate, but not neutral paraoxon and iso-OMPA. Asp-70 was also important for binding of positively charged pralidoxime (2-PAM) and for activation of re-activation by excess 2-PAM. Excess 2-PAM had an effect similar to substrate activation, suggesting the binding of 2 mol of 2-PAM to wild-type but not to the D70G mutant. A surprising result was that Asp-70 was important for irreversible aging, the D70G mutant having a 3-and 8-fold lower rate of aging for paraoxon-inhibited and di-isopropyl fluorophosphate-inhibited BuChE. Mutants of Asp-70 had the same rate constants for phosphorylation and re-activation by 2-PAM as wild-type. The A277W mutant behaved like wild-type in all assays. Our results predict that people with the atypical (D70G) variant of BuChE will be more sensitive to the toxic effects of echothiophate, but will be equally sensitive to paraoxon and di-isopropyl fluorophosphate. People with the D70G mutation will be resistant to re-activation of their inhibited BuChE by 2-PAM, but this will be offset by the lower rate of irreversible aging of inhibited BuChE, allowing some regeneration by spontaneous hydrolysis.

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“…(4) The upper pK calculated from the pH rate profile for dealkylation is consistent with that of histidine H + -440. (5) The Glu199Gln mutant of T. californica AChE [16], Glu202Gln mutant of mouse AChE [14], Glu202Gln mutant of human AChE [17,18], Trp86Ala and Phe330Ala mutants of human AChE [19,20] and the corresponding mutations in human butyrylcholinesterase [14,15,21] showed much diminished capacity for dealkylation when inhibited with soman. (6) 99 % of the product of the reaction is derived from a tertiary rather than a secondary carbenium ion [22].…”

Section: Methodsmentioning

confidence: 99%

Molecular dynamics study of active-site interactions with tetracoordinate transients in acetylcholinesterase and its mutants

Enyedy

Kovach

Bencsura

2001

Biochemical Journal

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The role of active-site residues in the dealkylation reaction in the P S C S diastereomer of 2-(3,3-dimethylbutyl)methylphosphonofluoridate (soman)-inhibited Torpedo californica acetylcholinesterase (AChE) was investigated by full-scale molecular dynamics simulations using CHARMM : 400 ps equilibration was followed by 150-200 ps production runs with the fully solvated tetracoordinate phosphonate adduct of the wild-type, Trp84Ala and Gly199Gln mutants of AChE. Parallel simulations were carried out with the tetrahedral intermediate formed between serine-200 Oγ of AChE and acetylcholine. We found that the NεH in histidine H + -440 is positioned to protonate the oxygen in choline and thus promote its departure. In contrast, NεH in histidine H + -440 is not aligned for a favourable proton transfer to the pinacolyl O to promote dealkylation, but electrostatic stabilization by histidine H + -440 of the developing anion on the phosphonate monoester occurs. Destabilizing interactions between residues and the alkyl fragment

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The Role of Glutamate-199 in the Aging of Cholinesterase

Cited by 54 publications

References 0 publications

Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6

Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6

Importance of aspartate-70 in organophosphate inhibition, oxime re-activation and aging of human butyrylcholinesterase

Molecular dynamics study of active-site interactions with tetracoordinate transients in acetylcholinesterase and its mutants

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