This brief review describes the reactions of organophosphorus compounds with cholinesterases, neuropathy target esterase, and phosphoric triester hydrolases with respect to their toxicity. It also describes antidotes, protectors, and decontaminating agents against organophosphates.U ovom prikazu opisane su reakcije organofosfornih (OP) spojeva s kolinesterazama, hidrolazama fosfornih triestera (PTH) i esterazom koja je povezana s neuropatskim djelovanjem OP spojeva (NTE). Kolinesteraze i NTE su serinske esteraze. Organofosforni spojevi inhibiraju te enzime zato što fosforiliraju serin u aktivnom mjestu enzima. Inhibicija acetilkolinesteraze (AChE) je glavni uzrok akutne toksičnosti OP spojeva. Suprotno serinskim esterazama, PTH enzimi hidroliziraju OP spojeve i time ih detoksiciraju. Oksimi su antidoti kod akutnog otrovanja OP spojevima zato što defosforiliraju inhibiranu AChE. Oksimi dodatno štite slobodnu AChE od fosforiliranja. Hidrolaze fosfornih triestera, kao i butirilkolinesteraza (BChE), služe i za dekontaminaciju od OP spojeva, a oba enzima istražuju se i kao mogući protektori protiv otrovanja OP spojevima. U prikazu su opisane i molekulske strukture AChE i PTH

Elsa Reiner

U ovom prikazu opisane su reakcije organofosfornih (OP) spojeva s kolinesterazama, hidrolazama fosfornih triestera (PTH) i esterazom koja je povezana s neuropatskim djelovanjem OP spojeva (NTE). Kolinesteraze i NTE su serinske esteraze. Organofosforni spojevi inhibiraju te enzime zato što fosforiliraju serin u aktivnom mjestu enzima. Inhibicija acetilkolinesteraze (AChE) je glavni uzrok akutne toksičnosti OP spojeva. Suprotno serinskim esterazama, PTH enzimi hidroliziraju OP spojeve i time ih detoksiciraju. Oksimi su antidoti kod akutnog otrovanja OP spojevima zato što defosforiliraju inhibiranu AChE. Oksimi dodatno štite slobodnu AChE od fosforiliranja. Hidrolaze fosfornih triestera, kao i butirilkolinesteraza (BChE), služe i za dekontaminaciju od OP spojeva, a oba enzima istražuju se i kao mogući protektori protiv otrovanja OP spojevima. U prikazu su opisane i molekulske strukture AChE i PTH.This brief review describes the reactions of organophosphorus compounds with cholinesterases, neuropathy target esterase, and phosphoric triester hydrolases with respect to their toxicity. It also describes antidotes, protectors, and decontaminating agents against organophosphates

Reiner, Elsa

Hrčak - Portal of scientific journals of Croatia

323Arh Hig Rada Toksikol 2001;52:323-331Two groups of esterases react with organophosphorus (OP) compounds: serine esterasesand phosphoric triester hydrolases (PTHs; EC 3.1.8). The acute toxicity of OP compoundsis primarily due to inhibition of acetylcholinesterase (AChE; EC 3.1.1.7). The delayedpolyneuropathy, induced by some OP compounds, is related to the inhibition of theneuropathy target esterase (NTE). So far, NTE has not been classified in the EnzymeNomenclature (1). AChE and NTE are both serine esterases. Unlike serine esterases, OPcompounds are not inhibitors, but substrates of phosphoric triester hydrolases (PTHs):paraoxonase (PON; EC 3.1.8.1) and DFPase (EC 3.1.8.2). The products of hydrolysis ofOP compounds are not toxic, and PTHs play an important role in OP detoxification.ORGANOPHOSPHORUSCOMPOUNDS ANDESTERASES: CURRENTRESEARCH TOPICSCONCERNING THETOXICITY OF ANDPROTECTION AGAINSTORGANOPHOSPHATESELSA REINERInstitute for Medical Research andOccupational Health, Zagreb, CroatiaReceived August 2001This brief review describes the reactions oforganophosphorus compounds withcholinesterases, neuropathy target esterase, andphosphoric triester hydrolases with respect to theirtoxicity. It also describes antidotes, protectors, anddecontaminating agents againstorganophosphates.Key words:acetylcholinesterase, butyrylcholinesterase,cholinesterases, DFPase, neuropathy target esterase,oximes, paraoxonase, phosphoric triester hydrolases,serine esterasesPresented as an invited State-of-the-Art Lecture at the Chemical and Biological Medical Treatment Symposium, 7-12 May 2000, Spiez,SwitzerlandREVIEW324The structural formulae of several OP compounds are given in Figure 1. Sarin, soman,tabun, and VX are nerve agents. Paraoxon and DFP are characteristic substrates of thePTHs. Both serine esterases and PTHs react with OP compounds on the same ester oranhydride bond marked in the figure by an undulated line.For detailed information, readers are invited to consult books and conferenceproceedings listed in references 2-12 at the end of this brief review. Reference 13summarises the early literature on the subject. Individual papers are not quoted in the text,because their number would grossly exceed the length of the review.REACTIONS OF OP COMPOUNDS WITH SERINE ESTERASESFigure 2 shows the reactions of serine esterases with OP compounds. The inhibition ofserine esterases is due to phosphorylation of the active-site serine. This reaction proceedsvia an intermediate Michaelis-type complex between the enzyme and OP. TheFigure 1 Structural formulae of several organophosphorus compoundsReiner E. ORGANOPHOSPHORUS COMPOUNDS AND ESTERASESArh Hig Rada Toksikol 2001;52:323-331.o325phosphorylated enzyme (EP) is catalytically inactive. Dephosphorylation with water(spontaneous reactivation) is very slow, while it is faster with oximes (oxime reactivation).Depending on the substituents on the phosphorus, EP can undergo dealkylation, which istermed ageing. An aged enzyme cannot be reactivated.Figure 2 Reactions of serine esterases with organophosphorus compoundsReiner E. ORGANOPHOSPHORUS COMPOUNDS AND ESTERASESArh Hig Rada Toksikol 2001;52:323-331326Acetylcholinesterase is a globular protein and its three-dimensional structure is known.Its physiological substrate is acetylcholine. Figure 3 shows a schematic drawing of AChEmolecule. The active site of AChE is in the centre of the molecule, accessible through anarrow gorge lined with water molecules. The catalytic triad (serine, histidine and glutamicacid), a choline-binding pocket, and an acyl-binding pocket form the active site.Furthermore, AChE has an allosteric site close to the rim of the gorge. The allosteric siteis catalytically inactive. However, reversible binding of substrates or other ligands to thatsite affects catalysis in the active site. The effect is usually inhibitory. There is some evidencethat the AChE molecule might have a Òback doorÓ through which products of substratehydrolysis leave the enzyme.Figure 3 Schematic drawing of the acetylcholinesterase molecule(prepared by Zoran Radiæ, UCSD, USA)Research on AChE is now directed towards identification of individual amino acidswhich participate in the individual steps of catalysis. The experimental approach consistsof preparing site-directed mutants, correlating the catalytic properties with the structureof the mutant and modelling the enzyme/ligand complexes. The results are expected tofurther elucidate the mechanism of action of AChE. This, in turn, might facilitate a moreReiner E. ORGANOPHOSPHORUS COMPOUNDS AND ESTERASESArh Hig Rada Toksikol 2001;52:323-331.o327rational design of compounds which would prevent phosphorylation of the active site byOPs, facilitate reactivation of EP and slow down ageing. The toxicity of OPs is primarilydetermined by these three reactions.NTE has only recently been cloned and shown to be unrelated to any known serinehydrolases. Its physiological role is not known. The OP compounds are inhibitors of NTE,but only some OPs cause delayed polyneuropathy. These OPs phosphorylate the active-site serine, which, in turn, is followed by ageing of the inhibited NTE. Polyneuropathy doesnot develop if ageing does not occur. This could indicate that the axonal maintenance issensitive to the negative charge on the aged NTE (Figure 2) and not to the activity of NTE.Phosphorylation and ageing of NTE occur within minutes, but degeneration of the longaxons takes weeks to develop.Present studies aim at clarifying the link between NTE and the OP-induced delayedpolyneuropathy. Studies are focused on the molecular structure of NTE and on compoundswhich promote OP-induced polyneuropathy without causing polyneuropathy themselves.The promoters known so far are NTE inhibitors, but it seems unlikely that NTE is thetarget enzyme.HYDROLYSIS OF OP COMPOUNDS BY PHOSPHORIC TRIESTERHYDROLASESThe mechanism of reaction of OP compounds with PTHs is different from the reaction ofOPs with serine esterases. The molecular structure of PTHs is also different. Phosphorictriester hydrolases require divalent cations for catalysis as shown in Figure 4. The cation(M++ in Figure 4) is embedded in histidine residues and binds the water molecule requiredfor OP hydrolysis. So far, nothing is known about the intermediate steps leading tohydrolysis, except for the kinetic evidence that a Michaelis complex is formed betweenthe enzyme and OP. The physiological role of PTHs is also not known.Figure 4 Reactions of phosphoric triester hydrolases withorganophosphorus compoundsReiner E. ORGANOPHOSPHORUS COMPOUNDS AND ESTERASESArh Hig Rada Toksikol 2001;52:323-331328Phosphoric triester hydrolases hydrolyse a broad range of OP compounds. Thesubstrate specificities of paraoxonase and DFPase are different, and the same holds forthe cation required for catalysis. Paraoxonase in mammalian sera requires calcium ions,while DFPase in microorganisms usually depends on magnesium, zinc, or manganesecations. Recently DFPase was also found in plants and its activity was stimulated bymanganese ions. Phosphoric triester hydrolases are stereoselective like AChE. The OPenantiomer which is more quickly hydrolysed by PTHs is less inhibitory for AChE, andvice versa. Phosphoric triester hydrolases have recently been shown to hydrolyse carboxylicacid esters such as phenylacetate, which is a characteristic substrate of arylesterases (EC3.1.1.2) (1).The search for natural substrate(s) and the physiological role of PTHs continues.Mammalian serum paraoxonases seem to act against cellular damage from toxic agentsand oxidised lipids, and there are some indications that human serum paraoxonases mightbe markers of lipid metabolism disorders. The polymorphism of these enzymes in humansera is well established. Phosphoric triester hydrolases have recently been cloned andmutants prepared. This has greatly enhanced studies on substrate specificity andmechanism of substrate hydrolysis. The in vivo role of PTHs in detoxification of OPs andtheir use in decontamination from OPs constitutes a very lively field of research.OXIMES AS ANTIDOTES AND PROTECTORS AGAINST OP COMPOUNDSThe primary mechanism of action of oximes as antidotes is to reactivate the phosphorylatedAChE (Figure 2). The pyridinium oximes PAM-2 and HI-6 (Figure 5) and the bis-pyridiniumdioxime toxogononin are therapeutic drugs in OP poisoning. Not a single oxime preparedso far is active against a very broad range of OPs. Moreover, none has been known to actagainst all four nerve agents. The search for better antidotes is still based on the trial-and-error approach. The same applies to oximes and other compounds used as prophylacticagents. Imidazolium oximes, and more recently quinuclidinium derivatives, are underevaluation. Two compounds from those groups are shown in Figure 5.The product of dephosphorylation by oximes is the phosphorylated oxime (Figure 2).Phosphorylated oximes are potent AChE inhibitors, but as they are very unstable,phosphorylation of the reactivated AChE by phosphorylated oximes has seldom beenreported.Oximes themselves are also toxic. They bind to AChE as reversible inhibitors.Reversible inhibitors form complexes with AChE either in the active site or in the allostericsite or in both sites of the enzyme. Reversible inhibitors, including oximes, protect AChEfrom phosphorylation. When the reversible inhibitor binds to the active site, the protectionis due to direct competition between the OP and reversible inhibitor. Binding of a reversibleinhibitor to the allosteric site induces indirect protection of the active site. This has beenwell documented by in vitro studies, but was less evaluated in experimental toxicology sofar.Reiner E. ORGANOPHOSPHORUS COMPOUNDS AND ESTERASESArh Hig Rada Toksikol 2001;52:323-331.o329Figure 5 Structural formulae of several oximesESTERASES AS ANTIDOTES, PROTECTORS AND DECONTAMINATINGAGENTS AGAINST OP COMPOUNDSPhosphorylation of serine esterases by OP compounds occurs on a 1:1 molar basis. Thisreaction inhibits the enzyme, but detoxifies OP. Phosphorylation of AChE causes toxicity,Reiner E. ORGANOPHOSPHORUS COMPOUNDS AND ESTERASESArh Hig Rada Toksikol 2001;52:323-331330because phosphorylated AChE cannot hydrolyse its physiological substrate acetylcholine.On the other hand, butyrylcholinesterase (BChE; EC 3.1.1.8) and carboxylesterase (EC3.1.1.1) in mammalian sera and tissues can be almost completely inhibited without anapparent toxic effect. This means that cholinesterases and carboxylesterases act asscavengers of OPs. However, once phosphorylated, the enzymes cannot detoxify anotherOP molecule unless there are oximes which would reactivate the enzyme. Consequently,cholinesterases and carboxylesterases act as stoichiometric scavengers, while combinedwith oximes they become catalytic scavengers.The feasibility of using serine esterases such as AChE and BChE combined withoximes as drugs against OPs has been demonstrated in rodents and in non-human primates.Protein engineering techniques have now enabled an intensive search for enzyme mutantswhich, ideally, should meet the requirement to react rapidly with OPs, to age slowly, andto be easily reactivated by oximes.PTHs also play a major role in the detoxification of OPs. The high toxicity of OPs foravian species has been attributed to the low activity of endogenous PTHs. Rabbits, whichhave a very high paraoxonase activity, are more resistant to paraoxon than other species.Human serum paraoxonases exhibit a substrate-dependent polymorphism, with low andhigh activity modes for each substrate. Analysis of polymorphisms in population groupsis suggested to identify individuals at risk. Studies on rodents have shown that administrationof purified paraoxonase significantly reduces the toxicity of paraoxon and other OPs. Thesepromising results stimulate present research on the use of PTHs and their mutants asdrugs against OPs.Finally, the use of esterases is suggested for the decontamination of skin, clothingand equipment. PTH from a microorganism immobilised on cotton wipes detoxifies nerveagents and OP pesticides. Purified AChE or BChE immobilised on polyurethane spongedo the same. Rinsing the sponge with oxime solutions restores the enzyme activity.Phosphoric triester hydrolases can be co-immobilised on the same sponge, increasing thecapacity of decontamination. Enzymes immobilised on matrices are more stable than insolution, and can be used repeatedly.REFERENCES1. Enzyme Nomenclature. Recommendations of the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes.San Diego (CA): Academic Press Inc.; 1992.2. Reiner E, Aldridge WN, Hoskin FCG, editors. Enzymes hydrolysing organophosphoruscompounds. Ellis Horwood Limited, Chichester; 1989.3. Massoulie J, Bacou F, Barnard E, Chatonnet A, Doctor BP, Quinn DM, editors. Cholinesterases:structure, function, mechanisms, genetics and cell biology. Conference Proceedings Series.Washington (DC): American Chemical Society; 1991.4. Reiner E, Lotti M, guest editors. Enzymes Interacting with organophosphorus compounds. ChemBiol Interact 1993;87:1-476.5. Mackness MI, Clerk M, editors. Esterases, lipases and phospholipases: From structure to clinicalsignificance. New York (NY): Plenum Press; 1994.Reiner E. ORGANOPHOSPHORUS COMPOUNDS AND ESTERASESArh Hig Rada Toksikol 2001;52:323-331.o3316. Quinn DM, Balasubramanian AS, Doctor BP, Taylor P, editors. Enzymes of the cholinesterasefamily. New York: Plenum Press; 1995.7. Price B, editor. Proceedings of the Second Chemical and Biological Medical TreatmentSymposium. Portland (ME): ASA; 1997.8. Doctor BP, Taylor P, Quinn DM, Rotundo RL, Gentry MK, editors. Structure and function ofcholinesterases and related proteins. New York (NY): Plenum Press; 1998.9. Reiner E, Simeon-Rudolf V, Doctor BP, Furlong CE, Johnson MK, Lotti M, et al, guest editors.Esterases reacting with organophosphorus compounds. Chem Biol Interact 1999;119/120:1-620.10. Bokan S, Orehovac Z, Price B, editors. Proceedings of the Chemical and Biological MedicalTreatment Symposium - Industry Portland (ME): ASA; 1999.11. Giacobini E, editor. Cholinesterases and cholinesterase inhibitors. London: Martin DunitzPublishers; 2000.12. Cousin X, Hotelier T, Chatonnet A. Esterases and alpha/beta hydrolase enzymes and relatives.[cited 28 Aug 2001]. Available from ESTHER wwwserver at URL: http://www.ensam.inra.fr/cgi-bin/ace/index13. Aldridge WN, Reiner E. Enzyme inhibitors as substrates. Interaction of esterases with esters oforganophosphorus and carbamic acids. Amsterdam: North-Holland Publishing Comp.; 1972.Sa¾etakORGANOFOSFORNI SPOJEVI I ESTERAZE: STANJE SADA'NJIHISTRAfiIVANJA TOKSI¨NOSTI I ZA'TITE OD ORGANOFOSFATAU ovom prikazu opisane su reakcije organofosfornih (OP) spojeva s kolinesterazama, hidrolazama fosfornih triestera (PTH) iesterazom koja je povezana s neuropatskim djelovanjem OP spojeva (NTE). Kolinesteraze i NTE su serinske esteraze.Organofosforni spojevi inhibiraju te enzime zato „to fosforiliraju serin u aktivnom mjestu enzima. Inhibicija acetilkolinesteraze(AChE) je glavni uzrok akutne  toksiŁnosti OP spojeva. Suprotno serinskim esterazama, PTH enzimi hidroliziraju OP spojeve i timeih detoksiciraju. Oksimi su antidoti kod akutnog otrovanja OP spojevima zato „to defosforiliraju inhibiranu AChE. Oksimi dodatno„tite slobodnu AChE od fosforiliranja. Hidrolaze fosfornih triestera, kao i butirilkolinesteraza (BChE), slu¾e i za dekontaminaciju odOP spojeva, a oba enzima istra¾uju se i kao moguæi protektori protiv otrovanja OP spojevima. U prikazu su opisane i molekulskestrukture AChE i PTH.KljuŁne rijeŁi:acetilkolinesteraza, butirilkolinesteraza, DFP-aza, esteraze fosfornih triestera, kolinesteraze, oksimi, paraoksonaza, serinskeesterazeReiner E. ORGANOPHOSPHORUS COMPOUNDS AND ESTERASESArh Hig Rada Toksikol 2001;52:323-331Requests for reprints:Elsa Reiner, Ph. D.Institute for Medical Research and Occupational HealthP. O. Box 291, HR-10001 Zagreb, CroatiaE-mail: ereiner@imi.hr

Organofosforni spojevi i esteraze: stanje sadašnjih istraživanja toksičnosti i zaštite od organofosfata

Abstract

Similar works

Full text

Available Versions

Hrčak - Portal of scientific journals of Croatia