Novel pyrene-containing organophosphates as fluorescent probes for studying aging-induced conformational changes in organophosphate-inhibited acetylcholinesterase

Amitai, Gabriel; Ashani, Yacov; Gafni, Ari; Silman, Israel

doi:10.1021/bi00538a013

Cited by 28 publications

(13 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Figure 9 displays the effect of two organophosphate (OP) inhibitors of AChE on the interaction of MB with Tc AChE. The two OP inhibitors shown in Scheme , paraoxon and pyrenebutyl ethyl phosphorofluoridate (PBEPF),40 both inhibit AChE irreversibly, by covalent reaction with its active‐site serine, S200; but the pyrenebutyl ethyl phosphoryl moiety of the conjugate produced by PBEPF is much bulkier than the diethylphosphoryl moiety in the conjugate obtained by reaction with paraoxon. Addition of the diethylphosphoryl‐ Tc AChE conjugate to MB produces a large “red” shift in its absorption maximum, to a value (∼678 nm) approaching that produced by the free enzyme; in contrast, addition of the PBEP‐ Tc AChE conjugate only slightly shifts the absorption maximum (∼667 nm), suggesting a substantial decrease in the affinity of MB for the PBEPF/ Tc AChE conjugate.…”

Section: Resultsmentioning

confidence: 99%

Structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo californica acetylcholinesterase

et al. 2012

View full text Add to dashboard Cite

The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic ''anionic'' subsite (CAS), the peripheral ''anionic'' subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both ''anionic'' subsites, but a single site is involved in inhibition. MB also quenches TcAChE's intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an~8°increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.

show abstract

Section: Resultsmentioning

confidence: 99%

Structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo californica acetylcholinesterase

et al. 2012

View full text Add to dashboard Cite

show abstract

“…Fluorescence decay of pyrenebutyl-containing organophosphates bound to acetylcholinesterase was found to have a lower quantum yield for non-aged conjugates than for aged ones [82]. This suggested that the pyrenebutyl group in aged conjugates is more deeply buried than in non-aged conjugates.…”

Section: Inhibition By Op and Aging Of The Inhibited Enzymementioning

confidence: 99%

Butyrylcholinesterase for protection from organophosphorus poisons: Catalytic complexities and hysteretic behavior

Masson

Lockridge

2010

Archives of Biochemistry and Biophysics

204

135

View full text Add to dashboard Cite

Butyrylcholinesterase is a promiscuous enzyme that displays complex kinetic behavior. It is toxicologically important because it detoxifies organophosphorus poisons (OP) by making a covalent bond with the OP. The OP and the butyrylcholinesterase are both inactivated in the process. Inactivation of butyrylcholinesterase has no adverse effects. However inactivation of acetylcholinesterase in nerve synapses can be lethal. OP-inhibited butyrylcholinesterase and acetylcholinesterase can be reactivated with oximes provided the OP has not aged. Strategies for preventing the toxicity of OP include a) treatment with an OP scavenger, b) reaction of nonaged enzyme with oximes, c) reactivation of aged enzyme, d) slowing down aging with peripheral site ligands, and e) design of mutants that rapidly hydrolyze OP. Option (a) has progressed through phase I clinical trials with human butyrylcholinesterase. Option (b) is in routine clinical use. The others are at the basic research level. Butyrylcholinesterase displays complex kinetic behavior including activation by positively charged esters, ability to hydrolyze amides, and a lag time (hysteresis) preceding hydrolysis of benzoylcholine and N-methyl indoxyl acetate. Mass spectrometry has identified new OP binding motifs on tyrosine and lysine in proteins that have no active site serine. It is proposed, but not yet proven, that low dose exposure involves OP modification of proteins that have no active site serine.

show abstract

“…It also originates in conformational changes, concomitant with the dealkylation reaction, at the enzyme active-site gorge. Several works showed that aging is associated with cholinesterase conformational change [14,24] and increase in conformational stability [25][26][27][28]. This might result from the formation of a salt bridge between catalytic protonated histidine and the negatively charged oxygen atom on the monophosphylate moiety.…”

Section: Scheme 1 the Mechanism Of Cholinesterase Phosphylation And Dmentioning

confidence: 99%

Aging of di-isopropyl-phosphorylated human butyrylcholinesterase

Masson

Fortier

Albaret

et al. 1997

Biochemical Journal

View full text Add to dashboard Cite

Organophosphate-inhibited cholinesterases can be reactivated by nucleophilic compounds. Sometimes phosphylated (phosphorylated or phosphonylated) cholinesterases become progressively refractory to reactivation; this can result from different reactions. The most frequent process, termed 'aging', involves the dealkylation of an alkoxy group on the phosphyl moiety through a carbocation mechanism. In attempting to determine the amino acid residues involved in the aging of butyrylcholinesterase (BuChE), the human BuChE gene was mutated at several positions corresponding to residues located at the rim of the active site gorge and in the vicinity of the active site. Mutant enzymes were expressed in Chinese hamster ovary cells. Wild-type BuChE and mutants were inhibited by di-isopropylfluorophosphate at pH 8.0 and 25 degrees C. Di-isopropyl-phosphorylated enzymes were incubated with the nucleophilic oxime 2-pyridine aldoxime methiodide and their reactivatability was determined. Reactivatability was expressed by the first-order rate constant of aging and/or the half-life of aging (t12). The t12 was found to be of the order of 60 min for wild-type BuChE. Mutations on Glu-197 increased t12 60-fold. Mutation W82A increased t12 13-fold. Mutation D70G increased t12 8-fold. Mutations in the vicinity of the active site serine residue had either moderate or no effect on aging; t12 was doubled for F329C and F329A, increased only 4-fold for the double mutant A328G+F329S, and no change was observed for the A328G mutant, indicating that the isopropoxy chain to be dealkylated does not directly interact with Ala-328 and Phe-329. These results were interpreted by molecular modelling of di-isopropylphosphorylated wild-type and mutant enzymes. Molecular dynamics simulations indicated that the isopropyl chain that is lost interacted with Trp-82, suggesting that Trp-82 has a role in stabilizing the carbonium ion that is released in the dealkylation step. This study emphasized the important role of the Glu-197 carboxylate in stabilizing the developing carbocation, and the allosteric control of the dealkylation reaction by Asp-70. Indeed, although Asp-70 does not interact with the phosphoryl moiety, mutation D70G affects the rate of aging. This indirect control was interpreted in terms of change in the conformational state of Trp-82 owing to internal motions of the Omega loop (Cys-65-Cys-92) in the mutant enzyme.

show abstract

Novel pyrene-containing organophosphates as fluorescent probes for studying aging-induced conformational changes in organophosphate-inhibited acetylcholinesterase

Cited by 28 publications

References 53 publications

Structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo californica acetylcholinesterase

Structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo californica acetylcholinesterase

Butyrylcholinesterase for protection from organophosphorus poisons: Catalytic complexities and hysteretic behavior

Aging of di-isopropyl-phosphorylated human butyrylcholinesterase

Contact Info

Product

Resources

About