Certain organophosphate (OP) cholinesterase inhibitors (ChEIs) are also known to bind to the muscarinic acetylcholine receptor (mAChR). The functional consequences of such binding were investigated here using the following OP compounds: VX, echothiophate, sarin, and soman. VX (charged at physiological pH) and echothiophate (formally charged) inhibited a specific signal transduction pathway in CHO cells expressing either the M(1) or M(3) mAChR. Hence, they blocked carbamylcholine (CCh)-induced cyclic adenosine monophosphate (cAMP) synthesis (muM) and had almost no effect on CCh-induced phosphoinositide (PI) hydrolysis. These substances were inactive on forskolin-induced cAMP inhibition signaling in CHO cells expressing M(2) mAChR. In binding studies, using [(3)H]-N-methyl scopolamine ([(3)H]NMS) as the competitor ligand, the ChEIs, VX and echothiophate exhibited binding to rat cortical mAChR with K(i) values in the muM range. The non-charged compounds, sarin and soman, were inert in modulating both cAMP metabolism and PI hydrolysis in CHO cells expressing M(1), M(2), and M(3) mAChRs, and no binding was observed in presence of [(3)H]NMS. These data suggest that VX and echothiophate act as function-specific blockers via a non-classical path of antagonistic activity, implying the involvement of allosteric/ectopic-binding site in M(1) and M(3) mAChRs. The functionally selective antagonistic behavior of echothiophate and VX makes them potential tools for dissecting the interactions of the mAChR with different G proteins.
The reactivity of human acetylcholinesterase (HuAChE) toward the chemical warfare agent VX [O-ethyl S-[2-(diisopropylamino)ethyl] methyl-phosphonothioate] and its stereoselectivity toward the P(S)-enantiomer were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere nc-VX [O-ethyl S-(3-isopropyl-4-methyl-pentyl) methylphosphonothioate]. Stereoselectivity of the wild-type HuAChE toward VX(S) is manifested by a 115-fold higher bimolecular rate constant (1.4 x 10(8) min(-1) M(-1)) as compared to that of VX(R). HuAChE was also 12,500-fold more reactive toward VX(S) than toward nc-VX(S), demonstrating the significance of the polar interactions of the ammonium substituent to their overall affinity toward VX. Indeed, substitution of the cation-binding subsite residue Trp86 by alanine resulted in a decrease of three orders of magnitude in HuAChE reactivity toward both VX enantiomers, with only a marginal effect on the reactivity toward the enantiomers of nc-VX. These results demonstrate that accommodation of the charged moieties of both VX enantiomers depends predominantly on interactions with the aromatic moiety of Trp86. Yet, these interactions seem to limit the stereoselectivity toward the P(S)-enantiomer, which for charged methylphosphonates is much lower than for the noncharged analogs, like sarin or soman. Marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 by asparagine (D74N) practically abolished stereoselectivity toward VX(S) (a 130-fold decrease), while substitution which retained the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers originates predominantly from a different orientation of the charged leaving group with respect to residue Asp74. Such different orientations of the charged leaving group in the HuAChE adducts of the VX enantiomers seem to be a consequence of intramolecular interactions with the bulky phosphorus alkoxy group.
Title: Poster (23) Phosphorylation and aging of AChE - insights from mutagenesis, mass spectrometry and structural studies Barak D, Ordentlich A, Kaplan D, Elhanani E, Segall Y, Barak R, Velan B, Shafferman A Ref: In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects, (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina:332, 2004 : PubMed
The origins of human acetylcholinesterase (HuAChE) reactivity toward the lethal chemical warfare agent O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX) and its stereoselectivity toward the P(S)-VX enantiomer (VX(S)) were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere O-ethyl S-(3-isopropyl-4-methylpentyl) methylphosphonothioate (nc-VX) as well as echothiophate and its noncharged analogue. Reactivity of wild-type HuAChE toward VX(S) was 115-fold higher than that toward VX(R), with bimolecular rate constants of 1.4 x 10(8) and 1.2 x 10(6) min(-1) M(-1). HuAChE was also 12500-fold more reactive toward VX(S) than toward nc-VX(S). Substitution of the cation binding subsite residue Trp86 with alanine resulted in a 3 order of magnitude decrease in HuAChE reactivity toward both VX enantiomers, while this replacement had an only marginal effect on the reactivity toward the enantiomers of nc-VX and the noncharged echothiophate. These results attest to the critical role played by Trp86 in accommodating the charged moieties of both VX enantiomers. A marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 with asparagine (D74N) practically abolished stereoselectivity toward VX(S) (130-fold decrease), while a substitution which retains the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers is mainly a result of a different interaction of the charged leaving group with Asp74.
Title: Major intermediates in organophosphate synthesis (PCl3, POCl3, PSCl3, and their diethyl esters) are anticholinesterase agents directly or on activation Segall Y, Quistad GB, Sparks SE, Casida JE Ref: Chemical Research in Toxicology, 16:350, 2003 : PubMed
Three phosphotrichlorides [phosphorus trichloride (PCl(3)), phosphorus oxychloride (POCl(3)), and thiophosphoryl chloride (PSCl(3))] with an annual U.S. production of >500,000,000 pounds and their diethyl esters are intermediates in the production of organophosphorus pesticides, plastics, flame retardants, and hydraulic fluids. They are classified as highly toxic to mammals based on acute oral and inhalation data with rats. This study considers their mechanisms of toxicity. PCl(3) and POCl(3) inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) from several species with in vitro IC(50) values of 5-36 and 88-1200 microM, respectively; PSCl(3) is a less potent inhibitor. These phosphotrichlorides have high vapor toxicity to houseflies with in vivo inhibition of brain AChE activity correlating with mortality. PCl(3) and POCl(3) produce cholinergic poisoning signs on ip administration to mice, and all three phosphotrichlorides give marked in vivo inhibition of serum BChE but not brain AChE activity. PCl(3) is a direct acting AChE inhibitor. Our earlier proposed activation of POCl(3) is confirmed here by preparing pure Cl(2)P(O)OH and its potassium and dicyclohexylamine salts that reproduce the action of POCl(3) as in vitro AChE inhibitors and toxicants in mice. PSCl(3) on hydrolysis yields Cl(2)P(O)SH [which oxidizes with peracid to Cl(2)P(O)SOH] as the proposed activation product. Vapors of (EtO)(2)PCl, (EtO)(2)P(O)Cl, and (EtO)(2)P(S)Cl are lethal to houseflies as in vivo AChE inhibitors, the first two acting directly and the last one on oxidative activation to (EtO)(2)P(O)Cl (possibly by P450) or (EtO)(2)P(O)SCl (a phosphorylating agent in a peracid oxidation system). Thus PCl(3), (EtO)(2)PCl, and (EtO)(2)P(O)Cl act directly as AChE inhibitors whereas POCl(3) and PSCl(3) undergo hydrolytic activation and (EtO)(2)P(S)Cl undergoes oxidative activation. In contrast, the toxicity to mice of phosphofluorides [FP(O)Cl(2), F(Cl)P(O)OH, and F(2)P(O)OH; studied as model compounds for comparison] may be due to liberating fluoride ion.
Title: Arachidonylsulfonyl derivatives as cannabinoid CB1 receptor and fatty acid amide hydrolase inhibitors Segall Y, Quistad GB, Nomura DK, Casida JE Ref: Bioorganic & Medicinal Chemistry Lett, 13:3301, 2003 : PubMed
Arachidonylsulfonyl fluoride (3), reported here for the first time, is similar in potency to its known methyl arachidonylfluorophosphonate (2) analogue as an inhibitor of mouse brain fatty acid amide hydrolase activity (IC(50) 0.1 nM) and cannabinoid CB1 agonist [3H]CP 55,940 binding (IC(50) 304-530 nM). Interestingly, 3 is much more selective than 2 as an inhibitor for fatty acid amide hydrolase relative to acetylcholinesterase, butyrylcholinesterase and neuropathy target esterase. N-(2-Hydroxyethyl)arachidonylsulfonamide (4) is at least 2500-fold less potent than N-(2-hydroxyethyl)arachidonamide (anandamide) (1) at the CB1 agonist site.
Title: Toxicological and structural features of organophosphorus and organosulfur cannabinoid CB1 receptor ligands Segall Y, Quistad GB, Sparks SE, Nomura DK, Casida JE Ref: Toxicol Sci, 76:131, 2003 : PubMed
Potent cannabinoid CB1 receptor ligands include anandamide [N-(2-hydroxyethyl)arachidonamide], Delta9-tetrahydrocannabinol, and 3H-CP 55,940 at the agonist site and selected organophosphorus esters (including some pesticides) and organosulfur compounds at a proposed closely coupled "nucleophilic" site. This study considers the toxicological and structural features of alkylfluorophosphonates, benzodioxaphosphorin oxides, alkanesulfonyl fluorides, and analogs acting at the nucleophilic site. Binding at the agonist site, using3H-CP 55,940 in assays with mouse brain membranes, is inhibited byO-isopropyl dodecylfluorophosphonate (compound 2), dodecanesulfonyl fluoride (compound 14) and dodecylbenzodioxaphosphorin oxide with IC50 values of 2-11 nM. Compounds 2 and 14 are also effectivein vivo, with 84% inhibition of mouse brain CB1 binding 4 h after intraperitoneal dosage at 30 mg/kg. Compound 14-inhibited CB1 in mouse brain requires about 3-4 days for recovery of 50% activity, suggesting covalent derivatization. Delayed toxicity (mortality in 0.3-5 days) from compounds 2, 14, and octanesulfonyl fluoride (18) is more closely associated with in vivo inhibition of brain neuropathy target esterase-lysophospholipase (NTE-LysoPLA) than with that of CB1 or acetylcholinesterase. NTE-LysoPLA inhibited by sulfonyl fluorides 14 and 18 cannot "age," a proposed requirement for NTE phosphorylated by organophosphorus-delayed neurotoxicants. Several octane- and dodecanesulfonamides with N-(2-hydroxyethyl) and other substituents based on anandamide give depressed mobility and recumbent posture in mice, but the effects do not correlate with potency for CB1 inhibition in vitro. Specific toxicological responses are not clearly associated with organophosphorus- or organosulfur-induced inhibition of the proposed CB1 nucleophilic site in mouse brain. On the other hand, the most potent CB1 inhibitors examined here are also NTE-LysoPLA inhibitors and cause delayed toxicity in mice.
Title: Selective inhibitors of fatty acid amide hydrolase relative to neuropathy target esterase and acetylcholinesterase: toxicological implications Quistad GB, Sparks SE, Segall Y, Nomura DK, Casida JE Ref: Toxicol Appl Pharmacol, 179:57, 2002 : PubMed
Fatty acid amide hydrolase (FAAH) plays an important role in nerve function by regulating the action of endocannabinoids (e.g., anandamide) and hydrolyzing a sleep-inducing factor (oleamide). Several organophosphorus pesticides and related compounds are shown in this study to be more potent in vivo inhibitors of mouse brain FAAH than neuropathy target esterase (NTE), raising the question of the potential toxicological relevance of FAAH inhibition. These FAAH-selective compounds include tribufos and (R)-octylbenzodioxaphosphorin oxide with delayed neurotoxic effects in mice and hens plus several organophosphorus pesticides (e.g., fenthion) implicated as delayed neurotoxicants in humans. The search for a highly potent and selective inhibitor for FAAH relative to NTE for use as a toxicological probe culminated in the discovery that octylsulfonyl fluoride inhibits FAAH by 50% at 2 nM in vitro and 0.2 mg/kg in vivo and NTE is at least 100-fold less sensitive in each case. More generally, the studies revealed 12 selective in vitro inhibitors for FAAH (mostly octylsulfonyl and octylphosphonyl derivatives) and 9 for NTE (mostly benzodioxaphosphorin oxides and organophosphorus fluoridates). The overall in vivo findings with 16 compounds indicate the expected association of AChE inhibition with acute or cholinergic syndrome and >70% brain NTE inhibition with delayed neurotoxic action. Surprisingly, 75-99% brain FAAH inhibition does not lead to any overt neurotoxicity or change in behavior (other than potentiation of exogenous anandamide action). Thus, FAAH inhibition in mouse brain does not appear to be a primary target for organophosphorus pesticide-induced neurotoxic action (cholinergic or intermediate syndrome or delayed neurotoxicity).
Binding of the endocannabinoid anandamide or of Delta(9)-tetrahydrocannabinol to the agonist site of the cannabinoid receptor (CB1) is commonly assayed with [3H]CP 55,940. Potent long-chain alkylfluorophosphonate inhibitors of agonist binding suggest an additional, important and closely-coupled nucleophilic site, possibly undergoing phosphorylation. We find that the CB1 receptor is also sensitive to inhibition in vitro and in vivo by several organophosphorus pesticides and analogs. Binding of [3H]CP 55,940 to mouse brain CB1 receptor in vitro is inhibited 50% by chlorpyrifos oxon at 14 nM, chlorpyrifos methyl oxon at 64 nM and paraoxon, diazoxon and dichlorvos at 1200-4200 nM. Some 15 other organophosphorus pesticides and analogs are less active in vitro. The plant defoliant tribufos inhibits CB1 in vivo, without cholinergic poisoning signs, by 50% at 50 mg/kg intraperitoneally with a recovery half-time of 3-4 days, indicating covalent derivatization. [3H-ethyl]Chlorpyrifos oxon may be suitable for radiolabeling and characterization of this proposed nucleophilic site.
Title: Resolving pathways of interaction of covalent inhibitors with the active site of acetylcholinesterases: maldi-tof/ms analysis of various nerve agent phosphyl adducts Elhanany E, Ordentlich A, Dgany O, Kaplan D, Segall Y, Barak R, Velan B, Shafferman A Ref: Chemical Research in Toxicology, 14:912, 2001 : PubMed
Understanding reaction pathways of phosphylation, reactivation, and "aging" of AChE with toxic organophosphate compounds is both a biochemical and a pharmacological challenge. Here we describe experiments which allowed to resolve some of the less well understood reaction pathways of phosphylation and "aging" of acetylcholinesterase (AChE) involving phosphoroamidates (P-N agents) such as tabun or the widely used pesticide methamidophos. Tryptic digests of phosphylated AChEs (from human and Torpedo californica), ZipTip peptide fractionation and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS) enabled reproducible signal enrichment of the isotopically resolved peaks of organophosphoroamidate conjugates of the AChE active site Ser peptides. For tabun and its hexadeuterio analogue, we find, as expected, that the two phosphoramidate adducts of the active site peptide differ by 6.05 mass units but following aging we find that the two corresponding phospho-peptides have identical molecular weights. We further show that the aging product of paraoxon-AChE adduct is identical to the aging product of the tabun-AChE conjugate. These results unequivocally demonstrate that the pathway of aging of tabun adducts of the human or the Torpedo californica AChEs proceeds through P-N bond scission. For methamidophos, we show that phosphylation of AChE involves elimination of the thiomethyl moiety and that the spontaneous reactivation of the resulting organophosphate adduct generates the phosphorus free AChE active site Ser-peptide.
Acetylcholinesterases (AChEs) form conjugates with certain highly toxic organophosphorus (OP) agents that become gradually resistant to reactivation. This phenomenon termed "aging" is a major factor limiting the effectiveness of therapy in certain cases of OP poisoning. While AChE adducts with phosphonates and phosphates are known to age through scission of the alkoxy C-O bond, the aging path for adducts with phosphoroamidates (P-N agents) like the nerve agent N,N-dimethylphosphonocyanoamidate (tabun) is not clear. Here we report that conjugates of tabun and of its butyl analogue (butyl-tabun) with the E202Q and F338A human AChEs (HuAChEs) age at similar rates to that of the wild-type enzyme. This is in marked contrast to the large effect of these substitutions on the aging of corresponding adducts with phosphates and phosphonates, suggesting that a different aging mechanism may be involved. Both tabun and butyl-tabun appear to be similarly accommodated in the active center, as suggested by molecular modeling and by kinetic studies of phosphylation and aging with a series of HuAChE mutants (E202Q, F338A, F295A, F297A, and F295L/F297V). Mass spectrometric analysis shows that HuAChE adduct formation with tabun and butyl-tabun occurs through loss of cyanide and that during the aging process both of these adducts show a mass decrease of 28 +/- 4 Da. Due to the nature of the alkoxy substituent, such mass decrease can be unequivocally assigned to loss of the dimethylamino group, at least for the butyl-tabun conjugate. This is the first demonstration that AChE adducts with toxic P-N agents can undergo aging through scission of the P-N bond.
Organophosphorus acid anhydride (OP) nerve agents are potent inhibitors which rapidly phosphonylate acetylcholinesterase (AChE) and then may undergo an internal dealkylation reaction (called "aging") to produce an OP-enzyme conjugate that cannot be reactivated. To understand the basis for irreversible inhibition, we solved the structures of aged conjugates obtained by reaction of Torpedo californica AChE (TcAChE) with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman) by X-ray crystallography to 2.3, 2.6, or 2.2 A resolution, respectively. The highest positive difference density peak corresponded to the OP phosphorus and was located within covalent bonding distance of the active-site serine (S200) in each structure. The OP-oxygen atoms were within hydrogen-bonding distance of four potential donors from catalytic subsites of the enzyme, suggesting that electrostatic forces significantly stabilize the aged enzyme. The active sites of aged sarin- and soman-TcAChE were essentially identical and provided structural models for the negatively charged, tetrahedral intermediate that occurs during deacylation with the natural substrate, acetylcholine. Phosphorylation with DFP caused an unexpected movement in the main chain of a loop that includes residues F288 and F290 of the TcAChE acyl pocket. This is the first major conformational change reported in the active site of any AChE-ligand complex, and it offers a structural explanation for the substrate selectivity of AChE.
The stereoselectivity of the phosphonylation reaction and the effects of adduct configuration on the aging process were examined for human acetylcholinesterase (HuAChE) and its selected active center mutants, using the four stereomers of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman). The reactivity of wild type HuAChE toward the PS-soman diastereomers was 4.0-7.5 x 10(4)-fold higher than that toward the PR-diastereomers. Aging of the PSCS-somanyl-HuAChE conjugate was also >1.6 x 10(4)-fold faster than that of the corresponding PRCS-somanyl adduct, as shown by both reactivation and electrospray mass spectrometry (ESI/MS) experiments. On the other hand, both processes exhibited very limited sensitivity to the chirality of the alkoxy group Calpha of either PS- or PR-diastereomers. These stereoselectivities presumably reflect the relative participation of the enzyme in stabilization of the Michaelis complexes and in dealkylation of the respective covalent conjugates, and therefore could be utilized for further probing of the HuAChE active center functional architecture. Reactivities of HuAChE enzymes carrying replacements at the acyl pocket (F295A, F297A, and F295L/F297V) indicate that stereoselectivity with respect to the soman phosphorus chirality depends on the structure of this binding subsite, but this stereoselectivity cannot be explained only by limitation in the capacity to accommodate the PR-diastereomers. In addition, these acyl pocket enzyme mutants display some (5-10-fold) preference for the PRCR-soman over the PRCS-stereomer, while reactivity of the hydrophobic pocket mutant enzyme W86F toward the PRCS-soman resembles that of the wild type HuAChE. Residue substitutions in the H-bond network (E202Q, E450A, Y133F, and Y133A) and the hydrophobic pocket (F338A, W86A, W86F, and Y337A) result in a limited stereoselectivity for the PSCS- over the PSCR-stereomer. Aging of the PS-somanyl conjugates with all the HuAChE mutant enzymes tested practically lacked stereoselectivity with respect to the Calpha of the alkoxy moiety. Thus, the inherent asymmetry of the active center does not seem to affect the rate-determining step of the dealkylation process, possibly because both the PSCS- and the PSCR-somanyl moieties yield the same carbocationic intermediate.
Title: The Aromatic Moiety at Position-86 of HuAChE Accelerate the Aging of Phosphonyl-AChE Conjugates through Cation- Interactions Barak D, Ordentlich A, Stein D, Segall Y, Velan B, Benschop HP, De Jong LP, Shafferman A Ref: In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases, (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp.:246, 1998 : PubMed
Title: ESMS as a Unique Tool for the Molecular Monitoring of Reactions between HuAChE and Various OP-Agents Ordentlich A, Barak R, Barak D, Fischer M, Benschop HP, De Jong LP, Segall Y, Velan B, Shafferman A Ref: In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases, (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp.:249, 1998 : PubMed
The contribution of the oxyanion hole to the functional architecture and to the hydrolytic efficiency of human acetylcholinesterase (HuAChE) was investigated through single replacements of its elements, residues Gly-121, Gly-122 and the adjacent residue Gly-120, by alanine. All three substitutions resulted in about 100-fold decrease of the bimolecular rate constants for hydrolysis of acetylthiocholine; however, whereas replacements of Gly-120 and Gly-121 affected only the turnover number, mutation of residue Gly-122 had an effect also on the Michaelis constant. The differential behavior of the G121A and G122A enzymes was manifested also toward the transition state analog m-(N,N, N-trimethylammonio)trifluoroacetophenone (TMTFA), organophosphorous inhibitors, carbamates, and toward selected noncovalent active center ligands. Reactivity of both mutants toward TMTFA was 2000-11, 000-fold lower than that of the wild type HuAChE; however, the G121A enzyme exhibited a rapid inhibition pattern, as opposed to the slow binding kinetics shown by the G122A enzyme. For both phosphates (diethyl phosphorofluoridate, diisopropyl phosphorofluoridate, and paraoxon) and phosphonates (sarin and soman), the decrease in inhibitory activity toward the G121A enzyme was very substantial (2000-6700-fold), irrespective of size of the alkoxy substituents on the phosphorus atom. On the other hand, for the G122A HuAChE the relative decline in reactivity toward phosphonates (500-460-fold) differed from that toward the phosphates (12-95-fold). Although formation of Michaelis complexes with substrates does not seem to involve significant interaction with the oxyanion hole, interactions with this motif are a major stabilizing element in accommodation of covalent inhibitors like organophosphates or carbamates. These observations and molecular modeling suggest that replacements of residues Gly-120 or Gly-121 by alanine alter the structure of the oxyanion hole motif, abolishing the H-bonding capacity of residue at position 121. These mutations weaken the interaction between HuAChE and the various ligands by 2.7-5.0 kcal/mol. In contrast, variations in reactivity due to replacement of residue Gly 122 seem to result from steric hindrance at the active center acyl pocket
While non-reactivability of cholinesterases from their phosphyl conjugates (aging) is attributed to an unimolecular process involving loss of alkyl group from the phosphyl moiety, no conclusive evidence is available that this is the only reaction path and involvement of other post-inhibitory processes cannot be ruled out. To address this issue, molecular masses of the bacterially expressed recombinant human acetylcholinesterase and of its conjugates with a homologous series of alkyl methylphosphonofluoridates, were measured by electrospray-ionization mass spectrometry (ESI-MS). The measured mass of the free enzyme was 64,700 Da (calculated 64,695 Da) and those of the methylphosphono-HuAChE adducts, bearing isopropyl, isobutyl, 1,2-dimethylpropyl and 1,2,2-trimethylpropyl substituents, were 64,820, 64,840, 64,852 and 64,860 Da, respectively. These values reflect both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent. The composition of these adducts change with time to yield a common product with molecular mass of 64,780 Da which is consistent with dealkylation of the phosphonyl moieties. Furthermore, in the case of 1,2-dimethylpropyl methylphosphono-HuAChE, the change in the molecular mass and the kinetics of non-reactivability appear to occur in parallel indicating that dealkylation is indeed the predominant molecular transformation leading to 'aging' of phosphonyl-AChE adducts.
The role of the functional architecture of human acetylcholinesterase (HuAChE) active center in facilitating reactions with organophosphorus inhibitors was examined by a combination of site-directed mutagenesis and kinetic studies of phosphorylation with organophosphates differing in size of their alkoxy substituents and in the nature of the leaving group. Replacements of residues Phe-295 and Phe-297, constituting the HuAChE acyl pocket, increase up to 80-fold the reactivity of the enzymes toward diisopropyl phosphorofluoridate, diethyl phosphorofluoridate, and p-nitrophenyl diethyl phosphate (paraoxon), indicating the role of this subsite in accommodating the phosphate alkoxy substituent. On the other hand, a decrease of up to 160-fold in reactivity was observed for enzymes carrying replacements of residues Tyr-133, Glu-202, and Glu-450, which are constituents of the hydrogen bond network in the HuAChE active center, which maintains its unique functional architecture. Replacement of residues Trp-86, Tyr-337, and Phe-338 in the alkoxy pocket affected reactivity toward diisopropyl phosphorofluoridate and paraoxon, but to a lesser extent that toward diethyl phosphorofluoridate, indicating that both the alkoxy substituent and the p-nitrophenoxy leaving group interact with this subsite. In all cases the effects on reactivity toward organophosphates, demonstrated in up to 10,000-fold differences in the values of bimolecular rate constants, were mainly a result of altered affinity of the HuAChE mutants, while the apparent first order rate constants of phosphorylation varied within a narrow range. This finding indicates that the main role of the functional architecture of HuAChE active center in phosphorylation is to facilitate the formation of enzyme-inhibitor Michaelis complexes and that this affinity, rather than the nucleophilic activity of the enzyme catalytic machinery, is a major determinant of HuAChE reactivity toward organophosphates.
Title: Amino Acids Determining Specificity to OP-Agents and Facilitating the Aging Process in Human Acetylcholinesterase Ordentlich A, Kronman C, Stein D, Ariel N, Reuveny S, Marcus D, Segall Y, Barak D, Velan B, Shafferman A Ref: In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases, (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp.:221, 1995 : PubMed
Title: Contribution of aromatic moieties of tyrosine 133 and of the anionic subsite tryptophan 86 to catalytic efficiency and allosteric modulation of acetylcholinesterase Ordentlich A, Barak D, Kronman C, Ariel N, Segall Y, Velan B, Shafferman A Ref: Journal of Biological Chemistry, 270:2082, 1995 : PubMed
Substitution of Trp-86, in the active center of human acetylcholinesterase (HuAChE), by aliphatic but not by aromatic residues resulted in a several thousandfold decrease in reactivity toward charged substrate and inhibitors but only a severalfold decrease for noncharged substrate and inhibitors. The W86A and W86E HuAChE enzymes exhibit at least a 100-fold increase in the Michaelis-Menten constant or 100-10,000-fold increase in inhibition constants toward various charged inhibitors, as compared to W86F HuAChE or the wild type enzyme. On the other hand, replacement of Glu-202, the only acidic residue proximal to the catalytic site, by glutamine resulted in a nonselective decrease in reactivity toward charged and noncharged substrates or inhibitors. Thus, the quaternary nitrogen groups of substrates and other active center ligands, are stabilized by cation-aromatic interaction with Trp-86 rather than by ionic interactions, while noncharged ligands appear to bind to distinct site(s) in HuAChE. Analysis of the Y133F and Y133A HuAChE mutated enzymes suggests that the highly conserved Tyr-133 plays a dual role in the active center: (a) its hydroxyl appears to maintain the functional orientation of Glu-202 by hydrogen bonding and (b) its aromatic moiety maintains the functional orientation of the anionic subsite Trp-86. In the absence of aromatic interactions between Tyr-133 and Trp-86, the tryptophan acquires a conformation that obstructs the active site leading, in the Y133A enzyme, to several hundredfold decrease in rates of catalysis, phosphorylation, or in affinity to reversible active site inhibitors. It is proposed that allosteric modulation of acetylcholinesterase activity, induced by binding to the peripheral anionic sites, proceeds through such conformational change of Trp-86 from a functional anionic subsite state to one that restricts access of substrates to the active center.
Rigid analogs of acetylcholine (ACh) were designed for selective actions at muscarinic receptor subtypes. AF102B, AF125, AF150 and AF151 are such rigid analogs of ACh. Whilst AF125 is an M2 > M1 agonist, AF102B, AF150 and AF151 are centrally active M1 agonists. AF102B has a unique agonistic profile showing, inter alia, only part of the M1 electrophysiology of ACh and unusual binding parameters to mAChRs. AF150 and AF151 are more efficacious agonists than AF102B for M1 AChRs in rat cortex and in CHO cells stably transfected with the m1 AChR subtype. In various animal models for Alzheimer's disease (AD) all three agonists (AF102B, AF150 and AF151), and in particular AF102B, exhibited positive effects on mnemonic processes and a wide safety margin. Such agonists, and especially AF102B, can be considered as a rational treatment strategy in AD. Here we review some current features of these compounds, which may be relevant to a rational treatment strategy in AD. Comparison is made, whenever possible, with some new and old muscarinic agonists.
Substrate specificity determinants of human acetylcholinesterase (HuAChE) were identified by combination of molecular modeling and kinetic studies with enzymes mutated in residues Trp-86, Trp-286, Phe-295, Phe-297, Tyr-337, and Phe-338. The substitution of Trp-86 by alanine resulted in a 660-fold decrease in affinity for acetythiocholine but had no effect on affinity for the isosteric uncharged substrate (3,3-dimethylbutylthioacetate). The results demonstrate that residue Trp-86 is the anionic site which binds, through cation-pi interactions, the quaternary ammonium of choline, and that of active center inhibitors such as edrophonium. The results also suggest that in the non-covalent complex, charged and uncharged substrates with a common acyl moiety (acetyl) bind to different molecular environments. The hydrophobic site for the alcoholic portion of the covalent adduct (tetrahedral intermediate) includes residues Trp-86, Tyr-337, and Phe-338, which operate through nonpolar and/or stacking interactions, depending on the substrate. Substrates containing choline but differing in the acyl moiety (acetyl, propyl, and butyryl) revealed that residues Phe-295 and Phe-297 determine substrate specificity of the acyl pocket for the covalent adducts. Phe-295 also determines substrate specificity in the non-covalent enzyme substrate complex and thus, the HuAChE F295A mutant exhibits over 130-fold increase in the apparent bimolecular rate constant for butyrylthiocholine compared with wild type enzyme. Reactivity toward specific butyrylcholinesterase inhibitors is similarly dependent on the nature of residues at positions 295 and 297. Amino acid Trp-286 at the rim of the active site "gorge" and Trp-86, in the active center, are essential elements in the mechanism of inhibition by propidium, a peripheral anionic site ligand. Molecular modeling and kinetic data suggest that a cross-talk between Trp-286 and Trp-86 can result in reorientation of Trp-86 which may then interfere with stabilization of substrate enzyme complexes. It is proposed that the conformational flexibility of aromatic residues generates a plasticity in the active center that contributes to the high efficiency of AChE and its ability to respond to external stimuli.
31P NMR spectroscopy of butyrylcholinesterase (BChE), acetylcholinesterase (AChE), and chymotrypsin (Cht) inhibited by pinacolyl methylphosphonofluoridate (soman), methylphosphonodifluoridate (MPDF), and diisopropyl phosphorofluoridate (DFP) allowed direct observation of the OP-linked moiety of aged (nonreactivatable) and nonaged organophosphorus (OP)-ChE conjugates. The 31P NMR chemical shifts of OP-ChE conjugates clearly demonstrated insertion of a P-O- bond into the active site of aged OP-ChE adducts. The OP moiety of nonaged OP-ChEs was shown to be uncharged. The OP-bound pinacolyl moiety of soman-inhibited and aged AChE was detached completely, whereas only partial dealkylation of the pinacolyl group was observed for soman-inhibited BChEs. This suggests that the latter enzyme reacted with the less active stereoisomer(s) of soman. In the case of soman-inhibited Cht, no dealkylation could be experimentally detected for any of the four stereoisomers of OP-Cht adducts. Results are consistent with the contention that the phenomenon of enzyme-catalyzed dealkylation of OP adducts of serine hydrolases strongly depends on the orientation of both the catalytic His and the carboxyl side chain of either Glu or Asp positioned next to the catalytic Ser. The denatured protein of aged OP-ChE or OP-Cht is a convenient leaving group in nucleophilic displacements of tetrahedral OP compounds despite the presence of a P-O- bond. This indicates that the unusual resistance to reactivation of the aged enzyme cannot be ascribed to simple electrostatic repulsion of an approaching nucleophile. The broadening of the 31P NMR signal of native OP-ChEs relative to that of OP-Cht is in agreement with the crystal structure of AChE, showing that the active site region of ChEs in solution resides in a deep, narrow gorge.
We demonstrate here the correlation between protection afforded by pretreatment alone with parathion hydrolase purified from Pseudomonas sp. against tabun toxicity in mice and the kinetic parameters which are assumed to determine the in vivo detoxification of tabun by the same enzyme. Results show that 15 and 22 micrograms of parathion hydrolase per animal conferred a protective ratio of 3.94 and 5.65 respectively, against tabun toxicity, without post-exposure treatment.
Title: Prophylaxis against organophosphate poisoning by an enzyme hydrolysing organophosphorus compounds in mice Ashani Y, Rothschild N, Segall Y, Levanon D, Raveh L Ref: Life Sciences, 49:367, 1991 : PubMed
Parathion hydrolase purified from Pseudomonas sp. was injected i.v. into mice to demonstrate the feasibility of using organophosphorus acid anhydride (OPA) hydrolases as pretreatment against organophosphates (OP) poisoning. Results show that exogenous administration of as low as 7 to 26 micrograms of parathion hydrolase conferred protection against challenge with multiple median lethal doses (LD50) of diethyl p-nitrophenyl phosphate (paraoxon; 3.8-7.3 x LD50) and diethylfluorophosphate (DEFP; 2.9 x LD50) without administration of supportive drugs. The extent of protection observed was consistent with blood-parathion hydrolase levels and the kinetic constants of the enzymatic hydrolysis of paraoxon and DEFP by parathion hydrolase. OPA hydrolases not only appear to be potential prophylactic drugs capable of increasing survival ratio following OP intoxication but also to alleviate post-exposure symptoms.
Homologous aged and nonaged fluorescent organophosphorus conjugates of alpha-chymotrypsin (Cht) were used in a comparative spectroscopic study of the conformation of their active sites, employing the pyrene group as the fluorescent probe. Steady-state fluorescence measurements showed that the quantum yield of the pyrene probe which is stoichiometrically attached to the active site is ca. 20% lower in the aged conjugate, pyrenebutyl-O-P(O)(O-)-Cht (PBP-Cht), than in the nonaged conjugate, pyrenebutyl-O-P-(O)(OC2H5)-Cht (PBEP-Cht). Furthermore, fluorescence decay data indicate that quenching is dynamic and is not caused by oxygen. These data, together with collisional quenching data, imply that quenching originates in an internal interaction of the fluorophore with a group within the protein. Thus, interaction of the pyrene moiety with the polypeptide chain is significantly stronger in the aged than in the nonaged conjugate, implying a different orientation of the fluorophore with respect to the protein. Circular dichroism measurements, which reflect the asymmetry of the bound pyrene in the ground state, as well as circularly polarized luminescence studies, which reflect its asymmetry in the excited state, also show that the relative configuration of the pyrene moiety and the polypeptide chain is significantly altered upon aging. Aged conjugates obtained by use of various fluorescenct organophosphates [pyrenebutyl-O-P(O)Cl2, pyrenebutyl-O-P(O)(p-nitrophenoxy)Cl, pyrenebutyl-O-P(O)(p-nitrophenoxy)2] exhibit similar spectroscopic features, thus substantiating the hypothesis that instantaneous aging, by use of pyrenebutyl-O-P(O)Cl2, and dynamic aging, by gradual removal of an aryloxy group, yield a similar product. This finding provides strong support for the formation of a P-O- moiety in the aged conjugates, since the only expected common product of the two processes is PB-O-P(O)(O-)-Cht. Formation of excimers of the pyrene-containing organophosphorylchymotrypsin conjugates at concentrations above 3 X 10(-6) M is also reported.
