The esterases: Perspectives and problems

Chem.-Biol. Interactions, 87 (1993) 5 - 1 3

5

Elsevier Scientific Publishers Ireland Ltd.

THE ESTERASES: PERSPECTIVES AND PROBLEMS

W.N. ALDRIDGE

Roberts Institute of Health and Safety, University of Surrey, Guildford, Surrey GU2 5XH (UK)

SUMMARY

Many proteins capable of hydrolysing esters are present in biological material of all kinds (microorganisms, plants, invertebrates and vertebrates). Some serve, as indicated by their substrate specificity and distribution within organisms, a defined biological function. However for most esterases a rather general substrate specificity is found indicating that they may have a broad biological function. Their properties will be briefly reviewed with particular emphasis on inhibitors. The mechanism of hydrolysis of esters by many carboxylesterases (Besterases) is well established largely due to the reaction of OP compounds with their catalytic centre. For others, such as enzymes hydrolysing (i) OP compounds and/or (ii) carboxyl esters which are not inhibited by a time and temperature dependent reaction by OP compounds, reaction mechanisms are still conjecture. The purpose of this presentation is to explore similarities and differences between the esterases and to discuss possible routes for progress in the Aesterase group.

Key words: A-esterases -- B-esterases -- Serine esterases -- Serine proteases -Cysteine proteases -- Delayed neuropathy -- Neuropathy target esterase -Aging of phosphylated esterases

ESTERS AND ESTERASES

At the last meeting of this series on esterases [1] there was considerable discussion about the nomenclature of esterases with the particular aim of improving the entries under hydrolases in the 'Enzyme Nomenclature: recommendations (1984)'. Since that time Drs. Reiner, Walker, Hoskins and I have continued the discussion and our recommendations for three entries will probably be accepted. During the course of these discussions the one statement on Correspondence to: W.N. Aldridge, Robens Institute of Health and Safety, University of Surrey, Guildford, Surrey GU2 5XH, UK. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

which we all could agree is that with our present state of knowledge is it very difficult to provide a rational system of nomenclature. The main reason is for some of the esterases defined by operational statements we still have no clear view either of mechanism or function. Thus, descriptions of esterases for nomenclature purposes will depend, even for many B-esterases (those inhibited by organophosphorus (OP) compounds in a progressive and temperature dependent reaction), on a composite of substrate specificity, properties and mechanisms. It has long been accepted that inhibition by OP compounds has provided a powerful tool for the separation of esterases into types. Substrates often lack sensitivity because of difficulties in the measurement of low hydrolysis rates. Acylating inhibitors on the other hand can be used over a large concentration range, inhibit in a reaction in which one molecule of enzyme is inactivated by one molecule of inhibitor and can be used in controlled experimental conditions so that accurate kinetic data may be obtained. The separation of esterases into types by the use of acylating inhibitors is very useful although it is clearly not acceptable for enzyme nomenclature which is concerned for the most part with individual enzymes, i.e., liver esterase is not under the same EC number as acetylcholinesterase and lipase. As a result of research over many years with many different enzymes which can hydrolyse esters we can now make rather firm decisions about the meaning of their sensitivity to inhibition by acylating compounds such as esters of organophosphorus and substituted carbamic acids and organosulphur compounds. Those substances which interact with B-esterases react in a way which is entirely analogous to the enzyme substrate interaction.

RIc(o)x R1R2p(o)x R1S(O2)X R1R2NC(O)X R1R2C(O2)X

Fatty acid esters, amino acid and peptide esters: triglycerides hydrolysed at water/lipid interface. Esters of phosphoric, phosphonic and phosphinic acids. Sulphonic acids -- X often, but not always fluorine. Esters of mono- and disubstituted carbamic acids. Esters of carbonic acids [56].

This conclusion is based on a consensus view of the properties of the interactions with the above compounds. Thus it cannot be concluded that substrates go through an acylated esterase intermediate step because the intermediate has not been isolated -- the same applies to the reaction of esterases with carbamates. It cannot be concluded that OP compounds are substrates for esterases because all the phosphylated intermediates have been shown to spontaneously reactivate. The consensus view is the sum total of the evidence involving a large number of esterases and inhibitors which allows us to state quite firmly that if several OP compounds inhibit the hydrolysis of a substrate in a progressive and temperature dependent reaction then the catalytic mechanism involves an intermediate step in which a serine moiety is acylated and subsequently hydrolysed. It seems that neuropathy target esterase (NTE) may almost certainly be added to the list [2]. B-esterases are therefore synonymous with serine esterases. However sensitivity to OP compounds is not always seen for hydrolytic enzymes which have an

acylated serine intermediate step. For example the hydrolysis of monoesters of phosphoric acid by E. coli alkaline phosphatase operates through a phosphorylserine intermediate step [3] but so far it has not been shown to be inhibited by OP compounds. This may be due to the need for negative charges on the substrate to gain access to the catalytic centre. Other esterases which are not inhibited by acylating inhibitors have been lumped together under the term A-esterases. In the original definition [4] an Aesterase was an enzymic hydrolysis of a simple ester which was not inhibited by diethyl 4-nitrophenylphosphate. Although this obviously should be widened to include other OP compounds, hydrolysis of the OP inhibitor should not be an obligatory requirement. From the discussion in 1988 [1] changing substrate patterns on purification raised considerable doubt about the validity of the conclusions from substrate competition experiments. The definition of A-esterases should only state, as originally, that they hydrolyse uncharged esters and they are not inhibited in a progressive reaction by OP compounds and other acylating inhibitors. This is a large group of esterases including those which hydrolyse carboxylic esters, carbamic esters and esters of phosphorus acids. The essential question we should address is whether this insensitivity to inhibition by OP compounds indicates that the catalytic mechanism does not operate through an acylated intermediate step. MECHANISM

Following the elucidation of the primary structures of several acetylcholinesterases [5] the solution to the three dimensional structure of Torpedo acetylcholinesterase [6] has now been published. The catalytic centre appears to be a triad of serine, histidine and glutamate. Of special interest is the finding that the binding of the quaternary trimethylammonium moeity of choline mainly involves the methyl groups and charge transfer from the aromatic residues in the essentially hydrophobic pocket. Access to the catalytic site seems to be via an essentially hydrophobic and aromatic channel [7,8]. The term 'anionic site' has therefore been clarified -- this was recently predicted on the basis of a comparison of different substrates [58] but such a view was suggested many years ago from experiments with 'carbon choline' esters [9]. Further extensions of these findings to other enzymes of the serine esterase class will no doubt confirm the essential features of the structure and mechanism of their catalytic centres but perhaps more importantly the related features which explain substrate specificity, allosteric sites and the structures which facilitate the rapid movement of substrates and products between the external mileau and the catalytic centre. The mechanism of any of the enzymes which hydrolyse OP compounds at a reasonable rate (to distinguish them from the action of serine esterases on them) is not known. In general they require metal ions such as calcium or manganese and are rather sensitive to inhibition by metallic salts such as mercury, organomercury, nickel and copper which have affinity for sulphydryl groups [10,11]. They do not appear to be inhibited by low concentrations of iodoacetate. With our pre-

sent state of knowledge it seems unlikely that the mechanism is through an acylated serine though it has not be rigorously excluded. The involvement of an acylated cysteine and a displacement reaction involving an activated water molecule are both possible [12]. At present the proteases may be the best models for the esterases. The following is the current classification of proteases or peptidases into groups by their sensitivity to inhibitors [13]: SerineCysteine-

AsparticMetalMetallo-

Esters of phosphorus, sulphur and carbamic acids [15], peptide halomethylketones [14]. Chloromethylketones [16], diazomethylketones [17-19], epoxides [20-21] and unsaturated ketones [16]; metals with affinity for SH. Acylated pentapeptides (pepstatins; [54,55]); diazoacetyl compounds [13] EDTA, etc. Hydroxamic acid derivatives [57].

If it is assumed that these hydrolyses of essentially hydrophobic and lipophilic substrates involves hydrophobic regions in the active centre as has been recently shown for acetylcholinesterase (see above and [6]) then any reagents used for groups catalytically active on uncharged carboxylic and phosphorous acid esters ought also to be hydrophobic. While the chloromethylketone derivatives of peptides, utilising the substrate specificity for access to the catalytic centre, are potent inhibitors they do not discriminate between the serine and cysteine proteases. In contrast the diazomethylketones, epoxides and unsaturated ketones have shown much greater selectivity for the cysteine proteases [16]. Even so there are still matters for argument about the detailed mechanisms in the serine and cysteinyl proteases [22]. It was tentatively suggested at our previous meeting [11] a search should be made for hydrophobic reagents which will react with A-esterases in a progressive and time dependent manner. Without such compounds it seems difficult to envisage progress in the development of testable hypotheses for the mechanism of action of the A-esterases and in deciding if different substrates are or are not hydrolysed by the same enzyme. THE AGING REACTION

Aging has been studied extensively in phosphorylated or phosphonylated cholinesterases and is defined as reactions which lead to the loss of one of the groups attached to the phosphorus atom in the phosphylated serine. Up to the present the groups released have been linked to the phosphorus atom by oxygen. A negative charge is produced by a PO- and for the cholinesterases this has been fully confirmed by chemical means. The formation of this negatively charged phosphorus-containing group attached to the serine can occur by at least two reactions -- one of which is probably chemical with the break in the P-O-R group between 0 and R and another in which the catalytic centre of the enzyme

acts on the P-O-R ester bond to break between the P and 0 [15,23]. For routine purposes the determination whether aging of phosphylated cholinesterases has taken place has depended on whether they can or cannot be reactivated by oximes (usually 2-PAM). Reactivation of the inhibited cholinesterases by oximes utilises the trimethylammonium binding site for the oxime to obtain access to the catalytic centre [15] and they are therefore rather selective. Another method more generally useful has been developed using a high concentration of potassium fluoride at pH 5.2 [24]. Aging occurs in many other phosphylated esterases. Of particular interest is the aging of NTE since the initiation of the development of delayed neuropathy is thought to depend on aging of the phosphylated NTE [25- 27]. The structure-activity relationships have largely been established using the fluoride method [26]. For the cholinesterases the R or OR groups are released into the medium but for NTE inhibited by two compounds, diisopropylphosphorofluoridate and di-n-pentyl-2,2-dichlorvinylphosphate, the leaving group has been shown to become attached to a neighbouring site [24,28]. It may be unwise to assume that the NTE reaction mechanism always involves a transfer of R to this site and for some of the active structures (causing delayed neuropathy) an OR group may be released [27] and not be attached. Perhaps the transfer to a neighbouring site in chicken brain microsomes is a 'biological accident' -- it would be interesting to know if it occurs in NTE from brains of other species. There is little evidence to consider that the mechanism(s) involved for aging for the cholinesterases and NTE are fundamentally different. There is much current discussion about the quantitative relationship between the degree of inhibition and/or aged NTE essential for the development of delayed neuropathy [29,31,32,41,42,52]. The mechanism of aging of NTE after inhibition by a wide range of OP structures together with chemical proof that the 'fluoride method' always indicates that aging has taken place requires more attention. DISTRIBUTION AND FUNCTION

A particular distribution may lead to hypotheses about function. Great progress has been made in elucidating the function of the proteases [13,34 - 37]. One method used has been to test the sensitivity of biological systems to organophosphorus compounds (usually diisopropylphosphorofluoridate although others may well be better). Selective distribution and ease of isolation has also helped in the determination of the function of the cysteine proteases. Relatively recent examples of such an approach suggest that trypsin-like proteases are involved in transmembrane signalling in cytotoxic T cells [38], in the induction of DNA synthesis by cytoplasmic factors [39] and in the disposal of neurofilaments at nerve synapses [59]. Except for acetylcholinesterase the function of the esterases is much less well developed. No doubt the microsomal esterases in liver and intestines are a protective mechanism to convert to more water soluble compounds the huge variety of esters consumed in mammalian diets. These enzymes are serine esterases. Research on delayed neuropathy caused by OP compounds has led to the discovery

10

HN3~'~NMe2 NH2 +

/ P~OMe -o~

~ 0

of neuropathy target esterase (NTE) [27], probably a serine esterase [2]. Whatever is the current view about the mechanism of the initiation and development of delayed neuropathy [29-31,33,40,43,53] it seems probable that NTE has a function in the maintenance and/or repair of nerve axons. We have few hypotheses about the function of the A-esterases group of esterases. There can be little doubt that there are many different esterases in this group which hydrolyse OP compounds, carbamates and/or carboxylic esters. The distribution and concentration of the esterases which hydrolyse diethyl 4nitrophenylphosphate (paraoxon) and diethyl 2-isopropyl-6-methylpyrimidin-4-yl phosphate (diazoxon) and are found in the lipoprotein fraction of sheep and human serum may provides hints about a possible function [44-46]. The presence of enzymes hydrolysing OP compounds decreases their toxicity [47]. Until recently, it was thought that esterase inhibiting OP compounds were manmade chemicals with no known 'natural' structures. However recent work has identified a toxin produced under certain conditions by cyanobacteria as an OP compound which inhibits acetylcholinesterase by a progressive reaction and is lethal to mice with the usual signs of anticholinesterase poisoning after an intraperitoneal dose of approximately 20 ~g/kg body weight [48-50]. This a similar toxicity to the nerve gas soman. The structure has been elucidated [51]. Whether this breakthrough has any significance in the metabolism of higher organisms cannot be predicted at the present time. CONCLUSIONS

Progress in understanding the mechanism of the A-esterase group of enzymes will depend on research on the isolation, purification [61] and detailed examination of substrate specificity and properties of 'purified' proteins. It may be that some of the many hydrolyses of esters in mammalian and tissues of other species may be the same proteins as those acting as proteases in functioning biological systems. Progress would no doubt be accelerated if inhibitors were found capable of reacting with the groups essential for catalytic activity. With such inhibitors whether a group of substrates are or are not hydrolysed by the same enzyme can usually be established in impure preparations [60].

11 REFERENCES 1 E. Reiner, W.N. Aldridge and F.G. Hoskin (Eds.), Enzymes hydrolysing organophosphorus compounds, Ellis Horwood Ltd, Chichester, 1989, pp. 1-263. 2 P. Glynn, M. Ruffer-Turner, D. Read, S. Wylie and M.K. Johnson, Proteolytic cleavage of neuropathy target esterase, Toxicologist, Abstract No. 64. 12, (1992) 41. 3 W.N. Aldridge, T.E. Barman and H. Gutfreund, The rate of formation and decomposition of phosphoryl-phosphatase (Escherichia coli), Biochem. J., 92 (1964) 23C-25C. 4 W.N. Aldridge, Serum esterases (1) Two types of esterase A and B) hydrolysing p-nitrophenyl acetate, propionate and butyrrate, and a method for their determination, Biochem. J., 53 (1953) 110-117. 5 K. MacPhee-Quigley, P. Taylor and S. Taylor, Primary structures of the catalytic subunits from two molecular forms of acetylcholinesterase: a comparison of NH2-terminal and active centre sequences, J. Biol. Chem, 260 (1985) 12185-12189. 6 J.L. Sussman, M. Harel, F. Frolow, C. Oefner, A. Goldman, L. Toker and I. Silman, Atomic structure of acetylcholinesterase from Torpedo californica: a prototype acetylcholine-binding protein, Science, 253 (1991) 872-879. 7 A. Maelicke, Acetylcholine esterase: the structure, Trends Biochem. Sci., 16 (1991) 355-356. 8 F. Hucho, J. Jarv and C. Weise, Substrate binding sites in acetylcholinesterase, Trends Pharmacol. Sci., 12 (1991) 422-426. 9 V.P. Whittaker, Specificity, mode of action and distribution of cholinesterase, Physiol. Rev., 31 (1951) 312-343. 10 W.N. Aldridge, Serum esterases (2) An enzyme hydrolysing diethyl p-nitrophenyl phosphate (E600) and its identity with the A-esterase of mammalian sera, Biochem. J., 53 (1953) 117-124. 11 W.N. Aldridge, A-esterases and B-esterases in perspective, in: E. Reiner, W.N. Aldridge and F.C.G. Hoskin (Eds.), Enzymes hydrolysing organphosphorus compounds, Ellis Horwood Ltd, Chichester, 1989, pp. 1-14. 12 J. Jarv, Insight into the putative mechanism of esterase acting simultaneously on carboxyl and phosphoryl compounds, in: E. Reiner, W.N. Aldridge and F.C.G. Hoskin (Eds.), Enzymes hydrolysing organophosphorus compounds, Ellis Horwood Ltd, Chichester, 1989, pp. 221 - 225. 13 J.S. Bond and P.E. Butler, Intracellular proteases, Ann. Rev. Biochem., 56 (1987) 333-364. 14 E.N. Shaw, Design of irreversible inhibitors, in: M. Sandler (Ed.), Enzyme inhibitors as drugs, Macmillan Press Ltd, London, 1980, pp. 25-42. 15 W.N. Aldridge and E. Reiner, Enzyme inhibitors as substrates: interaction of esterases with esters of organophosphorus and carbamic acids, North-Holland Publishing Co., Amsterdam, 1972, pp. 1-328. 16 E. Shaw, Active-site-directed irreversible inhibitors, in: M. Sandler and H.J. Smith (Eds.), Design of enzyme inhibitors as drugs, Oxford University Press, Oxford, 1989, pp. 49-69. 17 R. Leary, D. Larsen, H. Watanabe and E. Shaw, Diazomethyl ketone substrate derivatives as active-site-directed inhibitors of thiol proteases, Biochemistry, 16 (1977) 5857- 5861. 18 G.D.J. Green and E. Shaw, Peptidyl diazomethyl ketones are specific inactivators of thiol proteinases, J. Biol. Chem., 256 (1981) 1923-1928. 19 E. Shaw, P. Wikstrom and J. Ruscica, An exploration of the primary specificity site of cathepsin B, Arch. Biochem. Biophys., 222 (1983) 424-429. 20 A.J. Barrett, A.A. Kembhavi, M.A. Brown, H. Kirschke, C.G. Knight, M. Tamai and K. Hanada, L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L, Biochem. J., 210 (1982) 189- 198. 21 C. Parkes, A.A. Kembhavi and A.J. Barrett, Calpain inhibition by peptide epoxides, Biochem. J., 230 (1985) 509-516. 22 L. Polgar and P. Halasz, Current problems in mechanistic studies of serine and cysteine proteases, Biochem. J., 207 (1982) 1-10. 23 J.W. Hovanec and C.N. Lieske, Spontaneous reactivation of acetylcholinesterase inhibited with para-substituted phenyl methylphosphonochloridate, Biochemistry, 11 (1972) 1051-1056.

12 24 B. Clothier and M.K. Johnson, Rapid aging of neurotoxic esterase after inhibition by diisopropyl phosphorofluoridate, Biochem. J., 177 (1979) 549-558. 25 M.K. Johnson, The delayed neuropathy caused by some organphosphorus esters: mechanism and challenge, Crit. Rev. Toxicol., 3 (1975) 289-316. 26 M.K. Johnson, Organophosphorus esters causing delayed neurotoxic effects: mechanism of action and structure/activity studies, Arch. Toxicol., 34 (1975) 259-288. 27 M.K. Johnson, The target for initiation of delayed neurotoxicity by organophosphorus esters: biochemical studies and toxicological applications, Rev. Biochem. Toxicol., 4 (1982) 141-212. 28 B. Clothier and M.K. Johnson, Reactivation and aging of neurotoxic esterase inhibited by a variety of organophosphorus esters, Biochem. J., 185 (1980) 739 - 747. 29 M. Lotti, S. Caroldi, E. Capodicasa and A. Moretto, Promotion of organophosphate-induced delayed polyneuropathy by phenylmethanesulfonyl fluoride, Toxicol. Appl. Pharmacol., 108 (1991) 234-241. 30 M. Lotti, The pathogenesis of organphosphate polyneuropathy, Crit. Rev. Toxicol., 21 (1995) 465 - 487. 31 C.N. Pope and S. Padilla, Potentiation of organophosphorus-induced delayed neurotoxicity by phenylmethylsulfonyl fluoride, J. Toxicol. Environ. Health, 31 (1990) 261- 273. 32 M.K. Johnson and J.M. Sail, Organophosphoramidation of neuropathy target esterase (NTE) is sufficient to initiate delayed neuropathy (DN) without the necessity of an 'aging' reaction, Toxicologist, 12, Abstract 63 (1992) p. 40. 33 A. Moretto, E. Capodicasa and M. Lotti, The clinical expression of organophosphate induced polyneuropathy (OPIDP) in rats is age dependent, Toxicologist, 12, Abstract 67 (1992) p. 41. 34 J. Sturzebecher, Inhibitors of thrombin, in: M. Markovitch (Ed.), The thrombin, CRC Press, Boca Ratan, 1984, pp. 131-160. 35 F. Mackwardt and J. Sturzebecher, Inhibitors of trypsin and trypsin-like enzymes with a physiological role, in: M. Sandler and H.J. Smith (Eds.), Design of inhibitors as drugs, Oxford Univeristy Press, Oxford, 1989, pp. 619-649. 36 M. Sandler (Ed.), Enzyme inhibitors as drugs, Macmillan Press Ltd, London, 1980, pp. 1- 285. 37 M. Sandler and H.J. Smith (Eds.), Design of enzyme inhibitors as drugs, Oxford University Press, Oxford, 1989, pp. 1-810. 38 N. Utsunomiya and N. Nakamshi, A serine protease triggers the initial step of transmembrane signalling in cytotoxic T cells, J. Biol. Chem., 261 (1986) 16514-16517. 39 R.L. Wong, J.K. Gutowski, M. Katz, R.H. Goldfarb and S. Cohen, Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors, Proc. Nat. Acad. Sci. U.S.A., 84 (1987) 241-245. 40 E. Capodicasa, M.L. Scapellato, A. Moretto, S. Caroldi and M. Lotti, Chlorpyrifos-induced delayed polyneuropathy, Arch. Toxicol., 65 (1991) 150-155. 41 M. Bertolazzi, S. Caroldi, A. Moretto and M. Lotti, Interaction of methamidophos with hen and human acetylcholinesterase and neuropathy target esterase, Arch. Toxicol., 65 (1991) 580 - 585. 42 E. Vilanova, M.K. Johnson and J.L. Vicedo, Interaction of some unsubstituted phosphoramidate analogs of methamidophos (O,S-dimethylphosphoroamidothioate) with acetylcholinesterase and neuropathy target esterase of hen brain, Pest. Biochem. Physiol., 28 (1987) 224 - 238. 43 A. Moretto, E. Capodicasa, M. Peraica and M. Lotti, Age sensitivity to organophosphateinduced delayed polyneuropathy: biochemical and biological studies in developing chicks, Biochem. Pharmacol., 10 (1991) 1497-1504. 44 M.I.Mackness, Possible medical significance of human serum 'A'-esterases, in: E. Reiner, W.N. Aldridge and F.C.G. Hoskin (Eds.), Enzymes hydrolysing organophosphorus compounds, Ellis Horwood Ltd, Chichester, 1989, pp. 202-213. 45 M.I. Mackness and C.H. Walker, Partial purification and properties of sheep serum 'A'esterases, Biochem. Pharmacol., 32 (1983) 2291-2296. 46 M.I. Mackness, S.D. Hallam and C.H. Walker, 'A'-esterase activity in the lipoprotein fraction of sheep and human serum, Biochem. Soc. Trans., 13 (1985) 135-136. 47 C.H. Walker and M.I. Mackness, 'A'-esterases and their role in regulating the toxicity of organophosphates, Arch. Toxicol., 60 (1987) 30-33.

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51 52

53

54

55

56 57 58

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W.O. Cook, J.A. Dellinger, S.S. Suigh, A.M. Dahlem, W.W. Carmichael and B.R. Beasley, Regional brain cholinesterase activity in rats injected intraperitoneally with anatoxin-a(s) or paraoxon, Toxicol. Letts., 49 (1989) 29-34. N.A. Mahmood and W.W. Carmichael, Anatoxin-a(s), an anticholinesterase from the cyanobacterium Anabaenaflos-Aqua NRC-525-17, Toxicon., 25 (1987) 1221-1227. E.G. Hyde and W.W. Carmichael, Anatoxin-A(S), a naturally occurring organophosphate, is an irreversible active site-directed inhibitor of acetylcholinesterase (EC 3.1.1.7), J. Biochem. Toxicol., 6 (1991) 195-201. S. Matsunaga, R.E. Moore, W.P. Niemczura and W.W. Carmichael, Anatoxin-a(s), a potent anticholinesterase from Anabaenaflos-aquae, J. Am. Chem. Soc., 111 (1989) 8021-8023. M.K. Johnson, E. Vilanova and D.J. Reed, Anomalous biochemical responses in tests of the delayed neuropathic potential of methamidophos (O,S-dimethyl phosphorothioamidate) its resolved isomers and some higher O-alkyl homologues, Arch. Toxicol., 65 (1991) 618-624. A. Moretto, M. Bertolazzi, E. Capodicasa, M. Periaca, R.J. Richardson, M.L. Scapellato and M. Lotti, Phenylmethanesulfonyl fluoride elicits and intensifies the clinical expression of neuropathic insults, Arch. Toxicol., 66 (1992) 67-72. J. Boger, M.S. Lohr, E.H. Ulm, M. Poe, E.H. Blain, G.M. Fanelli, T.Y. Lin, L.S. Payne, T.W. Schorn, B.l. LaMont, T.C. Vassil, I.I. Stabilito, D.F. Veber, D.H. Rich and A.S. Bopari, Novel renin inhibitors containing the amino acid statine, Nature (London). 303 (1983) 81-84. J. Boger, L.S. Payne, D.S. Perlow. N.S. Lohr, M. Poe, E.H. Blain, E.H. Ulm, T.W. Scborn, B.I. LaMont, T.Y. Lin, M. Kawai, D.H. Rich, and D.F. Veber, Renin inhibitors - - syntheses of subnanomolar, competitive, transition state analog inhibitors containing the novel analog of statine, J. Med. Chem., 28 (1985) 1779-1790. M.L. Bender and F.C. Wedler, Phosphate and carbonate ester 'aging' reactions with achymothrypsin: kinetics and mechanism, J. Am. Chem. Soc. 94 (1972) 2101-2109. M.A. Holmes and B.W. Matthews, Binding of hydroxamic acid inhibitors to crystalline thermolysin, Biochemistry, 20 (1982) 6912-6920. S.G. Cohen, E. Salih, M. Solomon, S. Howard, S.B. Chishti and J.B. Cohen, Reactions of l-bromo2-[14C] pinacolone with acetylcholinesterase from Torpedo nobiliana: effects of 5trimethylammonio-2-pentanone and diisopropyl fluorophosphate, Biochim. Biophys. Acta., 997 (1989) 167-175. B.I. Roots, Neurofilament accumulation induced in synapses by leupeptin, Science, 221 (1983) 971 - 972. W.N. Aldridge, A method for the characterisation of two similar B-esterases present in the chicken nervous system, Biochem. J., 93 (1964) 619-623. C. Hassett, R.J. Richter, R. Humbert, C. Chapline, J.W. Crabb, C.J. Omiecinski and C.E. Furlong, Characterisation of cDNA clones encoding rabbit and human paraoxonase: the mature protein retains its original sequence, Biochemistry, 30 (1991) 10141-10149.