PHARMACOLOGY
`Be THERAPEUTICS
`
`Volume 6, No.3
`
`1979
`
`C. HEIDELBERGER
`and D. H. KING
`
`W. E. STEWART II
`and L. S. LIN
`
`G. GABOR
`
`H. DENK
`
`Contents
`
`427 Triftuorothymidine
`
`.,
`443 Antivrial activities of intederons
`
`513 Management of cardiac arrhythmias occurring ~n
`myocardial infarction
`.
`
`551 Effect of detergents on the mixed function oxidase
`and other microsomal enzymes
`
`A.R;MAfN
`
`579 Mode of action of anticholinesterases
`
`i Erratum
`
`ISSN 0163-7258
`PHTHDI' 6(3) 4l7-628 (1979)
`
`ISBN 0 08 015 4837
`
`Printed in Great Britain by A. Wheaton. &. Co., Exeter
`
`45'
`
`j.
`
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`

`Pharmac. Thl!r. Vol. 6, pp. 579-628, 1979. Pergamon Pre •• Ltd. Prinled in Gr .... t Brilain.
`
`Specialist Subject Editor: FUMIO MATSUMURA
`
`MODE OF ACTION OF
`ANTICHOLINESTERASES
`
`A. R. MAIN
`Department of Biochemistry, North Cal'olina State University, Raleigh, North Cal'olina 27650
`
`1. INTRODUCTION
`The anticholinesterases of interest in this paper are the organophosphate and
`carbamate compounds employed as insecticides. In this capacity they act as nerve
`poisons by inhibiting cholinesterases (ChE's) essential to the.Qperation of certain vital
`nerv~s. Such nerves employ acetylcholine as their transmitter substance and are
`referred to as cholinergic neurons to distinguish them from nerves using different
`transmitters. The acetylcholine transmits nerve impulses by diffusion across the 20 nm
`synaptic gap separating a nerve from the muscle or gland it controls or from another
`nerve. The assigned function of the. essential ChE is to catalyze the hydrolysis of
`acetylcholine in the synaptic gap. The products of hydrolysis are 100,000 times less
`effective as transmitters than is acetylcholine. Thus. the ChB acts to control trans(cid:173)
`mission of nerve impulses by modulating the concentration of acetylcholine in the
`synaptic gap, particularly in the region of the post-synaptic membrane where ChB's
`. and cholinoreceptors appear to be principally located (Koelle, 1963; Koelle et al.,
`1974). When ChB is inhibited by organophosphate or carbamate compounds, the
`acetylcholine accumulates and its concentration remains at levels which are con(cid:173)
`tinuously too ·high to operate as signals. The cholinoreceptors are then saturated with
`acetylcholine, making the nerve system inoperative. A vital function controlled by the
`nerves fails and the animal dies.
`In mammals the respiratory system is thought to fail· first, and the animal dies of
`asphyxiation. According to O'Brien (1976) t\othing is known about the chain of events
`leading to the death of insects following inhibition of ChE.
`The function assigned to ChE is based on mammalian motor neurons which are the
`best understood of the cholinergic neurons. In the central nervous system of both
`mammals and insects the function of the . ChB is less certain. In mammals the
`peripheral neurons of the voluntary and parasympathetic systems are cholinergic,but
`in inse.cts the corresponding peripheral neurons use other transmitters and are not
`cholinergic. The central nervous system of insects does, however, contain cholinergic
`neurons and presumably the ChE's of thes.e neurons must be the targets of: organo(cid:173)
`phosphate and carbamate inhibitors.
`One of the puzzling features. associated withChE's is their distribution. In addition
`to their location in the post.:.synaptic membrane, they are found in a n1,lmber of other
`places including the axon of the . nerve and in the sarcolemma and sarcoplasm of
`muscle tissue. ChE's are also.found in.a variety of non-neural tissues such as the
`plasma, erythrocytes and platelets of blood (Zajicek, 1957). Curioursly enough, their
`existence in these latter tissues is of interest to the toxicologist since these are the
`ChB activities usually monitored to follow poisoning. Except for the post-synaptic
`acetylcholinesterase (AChE), no function has been established for ChE's; and even in
`its synaptic locale, some workers question whether the function of the ChB is
`confined to simple hydrolysis (e.g. Lui and Mittag, 1974; Wurtzel, 1967). To appreciate
`what is known about the neural events following inhibition of ChE, the limitations
`imposed by the uncertainties concerning its function must be considered· within ~ the
`framework of a number of complex neural systems. A description of these systems in
`the detail required involves histochemical, electrophysiological and anatomical con-
`
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`A. R. MAlN
`
`580
`siderations which are clearly beyond the scope of a single article. Further description
`of the neural aspects are therefore omitted from this account. If an account of the
`mode of action of these insecticides is considered to extend to the therapeutic and
`prophylactic measures taken in connection with mammalian poisoning, then an
`understanding of neural events is desirable. However, a description of the basic mode
`of action of one of the more dramatic therapeutic measures is included in the present
`account since it primarily involves the regeneration of inhibited ChE and does not
`depend on other neural events.
`While acknowledging the importance of the neural aspects, the inhibition of ChE
`remains the key event in poisoning by organophosphate and carbamate insecticides,
`and it will be the principal subject of the present article. The fundamental mechanism
`by which inhibition occurs is understood and provides a foundation upon which a
`reasonably well ordered' account of the complex inhibition and substrate phenomena
`observed with ChB can be developed.
`The studies leading to the basic mechanism have been described by Florey and
`Michelson (1973) as 'one of the brilliant pages of molecular and biochemical phar(cid:173)
`macology', and they involved both substrate and inhibition reactions. ChB substrate
`reactions are therefore included when relevant, particularly in the section dealing with
`the basic mechanism. There are other reaSOns for including the substrate reactions.
`For example, the development of oxime reagents which regenerate phosphorylated
`ChE's and are incorporated in the structures of certain· carbamate insecticides
`followed primarily from the study of substrate reactions. More currently, the concepts
`developed to explain inhibition at high substrate concentrations are now applied to the
`inhibition reaction. In addition. ChE's are differentiated largely on the basis of their
`substrate specificities. But the most compelling reason is the fact that SUbstrates and
`inhibitors react with ChB's by precisely the same mecbanism. namely, the central
`mechanism referred to above.
`It is possible to give an account of the mode of action of organophosphate and
`carbamate insecticides without including the substrate reactions, and this is often
`done. The reason is that the primary model for carbamate and organophosphate
`inhibitors historically was not acetylcholine or any other substrate. The primary
`model for .carbamates was eserine (pbysostigmine). which occurs naturally and was
`known initially as a powerful poison. Organophosphates, on the otber hand, are purely
`the creation of man. Moreover, they were recognized as poisons and used as
`insecticides for a number of years before tbey were found to poison by inhibiting
`CbB. Thus. the development of both organophosphate and carbamate insecticides has
`depended only partly on rational biochemical principles. Chance observations
`shrewdly exploited and the exhaustive screening of thousands of compounds have
`also played an important part. But the search for pesticides has contributed little to
`the discoveries which led to the basic mechanisms by which their mode of action is
`explained, and this area is therefore of peripheral rather tban of central interest to this
`article.
`Another aspect of ChB's of relevance to both the mode of action and development
`of insecticides is the variability of ChE's and their wide distribution in the animal
`kingdom. That ehB's occur in many different tissues has already been mentioned.
`They also are found in most members of the animal kingdom, ranging from planaria to
`elephants. For example, the organophosphate, Ruelene. is used to control parasitiC
`worms in cattle. Presumably, it acts by inhibiting the ehE's of the worms and thus
`killing them. Moreover, the diversity of ChE's may be one of the factors contributing
`to the selectivity of certain insecticides.
`Given the diversity and number of inhibitors of ChB's, the numerous areas involved
`and the vast literature,' it is not siJrprising that various descriptions of the mode of
`action of anti-ChE's tend to differ significantly since there is much room for selection
`and emphasis. Even reading the literature is a daunting and at times exasperating task
`as evidenced, for example, by the comments of Aldridge and Reiner (1972) on this
`'flood of paper'. They write in part that, 'anyone undertaking the publication of
`
`',:';'
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`Mode of action of anticholinestcrascs
`
`581
`further material ought to justify why they think it necessary'. In an -earlier COn(cid:173)
`tribution on anti-ChE's to the IEPT series, Usdin (1970) included 130 books, reviews
`and monographs and some 1000 references to the original literature in his reference
`list. While impressive and useful, neither list is exhaustive and much has appeared
`since 1970. In this article the reference list is shorte( and thus more selective-and
`open to bias. A number of key papers from the original literature are included; but
`obviously many have been omitted, either through ignorance or to avoid the bewil(cid:173)
`derment and loss of focus brought on by too many references. The objective is to
`present a reasonably coherent account authenticated with adequate, but not exhaus(cid:173)
`tive, support from the literature.
`
`2. CLASSIFICATION AND SUBSTRATE SPECIFICITY OF ChE'S
`ChE's catalyze the hydrolysis of a wide variety of aliphatic and aromatic carboxylic
`and thiocarboxylic esters of the general formula,
`
`o
`II
`R,-C-O-R. and
`
`o
`II
`R,-C-S-R.
`
`However, the property that distinguishes ChE's from other esterases is their ability to
`hydrolyze choline esters and other esters in which the R2 group contains a positively
`charged nitrogen function. While it is true that the best substrates are often choline
`esters, some choline esters are not good substrates. For example, acetylcholinesterase
`(AChE) from human erythrocytes hydrolyzes acetylcholine as rapidly or more rapidly
`than any other substrate, but the hydrolysis of butyryleholine and benzoylcholine is
`negligible. On the other hand, AChE hydrolyzes 3: 3-dimethyl butylacetate, the carbon
`isostere of ACh, at 60 per cent the rate of ACh while phenyl acetate is hydrolyzed as
`
`0
`CH 3
`II
`I
`CH 3 CCH 2CH2---Q-C-CH3
`I
`CH3
`
`3 : 3-d Imethyl butylacetate
`
`aCetylcholine
`
`rapidly as ACh at saturation substrate concentrations (Adams and Whittaker, 1949;
`Mounter and Whittaker, 1953; Krupka, 1963). Some authors have questioned the use
`of the term 'cholinesterase' to apply to esterases which hydrolyze aliphatic or
`aromatic esters more readily than choline esters; but in view of the specificities just
`mentioned, such a distinction seems unwarranted. What is apparent is that esterases
`which hydrolyze choline esters, whatever the degree of their specificity in this regard,
`can readily be distinguished from the many esterases which do not hydrolyze choline
`esters. And this brings us to the thorny question of classifying ChE'~.
`In 1914 Dale suggested that an esterase might exist which hydrolyzed ACh, "and in
`1926 Loewi and Navratil demonstrated that such an enzyme did exist in the course of
`work which also showed ACh .to be a chemical" transmitter of nerve impulses.
`However, it was not until 1932 that Stedman, Stedman and Easson measured the rate
`of hydrolysis of ACh by chemical means and showed that the enzyme responsible was
`distinct from other esterases known at that time. The enzyme was called choline(cid:173)
`esterase, and it was found first in horse serum. Soon after. ChE's were located in the
`sera and red blood cells of other animals and in nerve tissue including parts of the
`brain. In 1940 Alles and Hawes compared the substrate kinetics of the ChE in human
`serum with that in human red blood cells and found them to be different. In addition,
`they found that while the erythrocyte ChE hydrolyzed acetyl-~-methyl choline at a
`significant rate, the serum ChE hardly touched it. But the most interesting difference
`
`~::
`
`C
`0
`.... m
`i.
`t Jg
`"t::
`m
`E
`Q)
`{', F
`,.
`~.
`:~ '"
`
`!pR
`
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`582
`
`A. R. MAIN
`
`0
`CH3
`CH,
`II
`I
`I
`.
`CH,-N-CH 2 CH-O-CCH 3
`I
`a
`(3
`CH.
`acetyl·,8·methyl choline
`
`was in the kinetics. With the erythrocyte ChE, the velocity (v) of the reaction
`increased initially as the substrate concentration [S] increased and finally reached a
`maximum as would be expected from Michaelis-Menten kinetics. However, as [S]
`was increased fUrther. v began to decrease. a phenomenon which is variously
`described as inhibition by excess substrate or inhibition at high substrate concen(cid:173)
`trations. For both theoretical and practical reasons, the relationship is often shown by
`ploUing, ~log [S] or p[S] against v. A typical plot is shown in Fig. l(a). In contrast to
`the behavior of the erythrocyte ChE, the v against [S] plots of the serum ChB did not
`show inhibition at high values of [S], and the reaction was at first believed to follow
`Michaelis-Menten kinetics-a belief which has led to serious misinterpretations to the
`present day. In fact, the velocity of serum ChE reactions increases faster than
`predicted by Michaelis-Menten kinetics at high values of [S], so one might say that
`serum ChE's are activated by excess substrate. The erythrocyte and serum ehE v
`against [SJ relationships are compared in Fig. l(a) and l(b) using different fUnctions.
`
`v
`
`V
`
`Vmo
`
`a
`
`\
`\
`\
`
`/I
`/1
`...
`~ I
`I
`./ I
`I
`.-
`• Ii
`3 pl$l2
`viIS!
`FIG. I(a). Typical v against p[SJ plots for AChE and BuChE. The substrate is acetylcholine.
`The precise shape and positioning of the curves will depend on salt concentration. pH and
`temperature, but the form of the curves will remain as shown.
`
`/
`
`0
`
`4
`
`0
`
`FlO. l(b). Typical v against v/[S] plots of AChE and BuChE. High concentrations of
`acetylcholine inhibit AChE but activate BuChE. V u is more than twice V .... with BuChE but
`is only O.IV.,," with AChE. A Vu will be obtained if E~ breaks down to yield proJucts.
`Current theory assumes E~ is an EAS complex, but an allosteric complex, SEA, should be
`considered (Section 9.2).
`
`Augustinsson (1948) applied a kinetic analysis developed by Haldane (1930) to the
`behavior of the erythrocyte ChE. Inhibition by excess substrate is assumed to involve
`formation of an E~ intermediate complex in addition to the usual ES complex of
`Michaelis and Menten. The reaction schemes were as follows:
`
`~
`c:
`9>
`o o
`
`"0
`'<
`::::I.
`~'
`~[:;
`
`K.
`k
`E+S~ES_E+P
`
`Ks,
`ES+S~ES2
`
`SCHEME I.
`
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`Mode of action of anticholinesterases
`
`583
`
`E is ChE, S is acetyl choline, (ACh) and the ES2 complex is unreactive. The equation
`derived from Scheme 1 is:
`
`V
`v = 1 + KJ[S] + [S]/K ••
`
`(I)
`
`Equation 1 predicts that the plot of p[S] against v will be symmetrical and bell shaped
`like that in Fig. l(a). The maximum v at the apex of the plot is not V, the
`Michaelis-Menten maximum v. Rather, it is VOl't or the optimum v. At vOpl there is a
`corresponding and particular substrate concentration, [S)Ol'l' V. K. and K •• can be
`evaluated from [Slept. which can be read from the experimental plot, by the following
`procedures. The first derivative of the reciprocal of Eqn. (1) is taken with respect to
`[S], as follows:
`
`When v = Vap," d(v-I)/d[S} = 0 and [S)oPt at this point is given by
`
`[SloPt = V(K.KsJ.
`
`(2)
`
`(3)
`
`The relationship of V to Vopt is then obtained by substituting the expression for [S]OPI in
`Eqn. (3) for [S] in Eqn. (1) from which
`
`V/VOPl = 1 + 2V(K./KaJ.
`
`(4)
`
`Estimation of V, K. and K.. are of course important since they characterize the
`substrate reaction and allow quantitative comparisons to be made with other sub(cid:173)
`strates.
`Following Alles and Hawes, the question arose as to the relationship of ChE's
`found in other tissues to the two ChE's of the blood. The initial findings indicated that
`the ChE in nerve tissue behaved like that of the erythrocyte while ChE's found in
`tissues such as the pancreas behaved like the serum enzyme. Mendel and Rudney
`(1943) observed that the erythrocyte ChE did not hydrolyze tributyrin and methyl
`butyrate while serum ChE did. From this they concluded, quite incorrectly,. that the
`erythrocyte ChE was 'specific' in the sense that it "hydrolyzed" only choline esters
`while the serUm enzyme was" non-specific. They therefore suggested the name
`psuedo-ChE for the serum enzyme, a name which Glick (1945)," Cohen et at. (1949)
`and others have objected to, but which some authors still use (e.g. Silver, 1974), and
`they referred to the erythrocyte ChE as the 'true' or 'specific' ChE. Nachmansohn
`(1944) and Cohen et al. (1949) observed that the nerve ChE's" hydrolyzed butyryl(cid:173)
`choline (BuCh) very slowly. This is in marked contrast to the serum ehE which
`hydrolyzed BuCh more rapidly than2ACh. For this and other reasons, Augustinsson
`and Nachmansohn (1949) suggested that th~ erythrocyte type of ChE's be named
`acetylcholine-esterase. SimilarlY, Sturge and Whittaker (1949) suggested that the
`erythrocyte enzyme be named aceto-cholinesterase, and they also named the serum
`enzyme butyro-cholinesterase. With slight modifications both names have been widely
`adopted to describe these two types of ChB. Thus, the erythrocyte type is now
`commonly called acetylcholinesterase (AChE) while the serum type is called butyryl(cid:173)
`cholinesterase (BuChE).
`The specificities are compared in Table 1. The three substrates following ACh in
`Table 1 are the ones chiefly used to distinguish between AChE and BuChE. Some of
`these differences do not apply to insect ChE's. For example, By head ChE hydrolyzes
`BuCh quite readily, but in some other respects behaves like mammalian AChE. The
`BuChE's in the sera of certain species such as rat and ducks is propionyl rather than
`butyryl specific. Rabbit serum contains both AChE and BuChE (Main et al., 1977) and
`so on. The point is that broad generalizations concerning the type of ChE in a given
`
`c
`o
`m
`~ (tJ
`E
`"," F
`<I)
`
`·.·I~"'"
`
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`

`584
`
`A. R. MAIN
`
`T ABI.R I. Specificities of Mammalian· AChE and BuChE Toward Selected Substrates
`
`Velocities are relative to ACh == 100 per cent. Values are based on saturation or optimal
`suhstrate concentrations. Interpretations shOUld be made with reservations since K ..
`values are omitted. However, the table illustrates the classical specificity differences of the
`two ChE·s.
`
`Substrates
`
`AChE
`
`Code for
`ref & source'" BuChE
`
`Code for
`ref & source'"
`
`Acetylcholine
`Butyrylcholine
`Benzoylcholine
`(D, L)Acetyl-~-methylcholine
`Propionylcholine
`Acetyl thiocholine
`(D, L)Acetyl-~-methylthiocholine
`L-( + )Acetyl-p-methylcholine
`D-( - )Acetyl-p-methYlcholine
`Phenyl acetate
`3: 3-Dimethyl butylacetate
`Triacetin
`Tributyrin
`
`100
`2
`1.5
`33
`87
`83
`82
`54
`0
`113
`60
`42
`2
`
`I{a)
`](a)
`I (a)
`I(a)
`4(b)
`S(c)
`7('1)
`7(1)
`4(b)
`I (a)
`l(a)
`I(a)
`
`100
`330
`35
`I
`170
`140
`50
`2
`0
`91
`35
`14
`45
`
`3(A)
`SeA)
`5(A)
`3(A)
`SeA)
`9(B)
`6(B)
`6(B)
`2(A)
`SeA)
`SeA)
`SeA)
`
`·With one exception. electric eel AChE.
`"Source code: (a) hUman erythrocyte AChE; (b) bovine erythrocyte AChE; (c) electric
`eel AChE; (A) human serum BuChE; (B) horse serum BuChE. Reference Code: 1. Adams.
`1949; 2. Mounter et al .• 1953; 3. Hastings, ]966; 4. Krupka, 1966; 5. Adams and Whittaker,
`1949; 6. Glick. 1938; 7. Beckett, 1967; 8. HeilbroDD, 1959; 9. Glick, 1939.
`
`tissue are usually not valid, and one cannot always predict the type of ChE by its
`location. Even nerve tissues contain both types of ChE. The end plates of various
`motor neurons in a number of mammals appear to contain as much or more BuChE
`than AChE as judged by histochemical techniques (Barnard, Rymaszewska and
`Wieckowski, 1971). An extreme example was the end plate of a motor neuron serving
`the sternomastoid muscle of the Rhesds monkey which contained 9 per cent AChE
`and 53 per cent BuChE. BuChE's predominated in all six monkey end plates
`examined. The available evidence suggests that BuChE's and' AChE's have much in
`common, and it is conceivable that they are variations on a theme possibly in the way
`of trypsin,chymotrypsin and elastase are related. Their moleCUlar weights fall in the
`same range, and they appear to have similar suhunit structures; but we still do not
`know enough about their chemical composition and physical structures to evaluate
`their fundamental relationship.
`Referring again to Table 1, BuChE readily hydrolyzes acetyl-J3-methyl thiocholine
`although acetyl-J3-methylcholine is hydrolyzed very slowly. Glick (1939) introduced
`the use of thiocholine esters and they have taken on a special significance in recent
`years because of the widely used method of Ellman et al. (1961) to determine ChE
`activities. The Ellman procedure is both specific for ehB's and has tbe convenience of
`spectrophotometric methods. It depends on the Ellman reagent, 5,5'-dithiobis(2-nitro(cid:173)
`benzoic acid) or DTNB, and the, use of thiocholine esters. Thiocholine esters are also
`used in histochemical staining techniques to locate ChE's in tissues (e.g. Koelle, 1963;
`Booth and Metcalf. 1970).
`
`3. THE CENTRAL REACTION SECHEME AND THE
`ACTIVE SITE OF ChE's
`Much of what is known about the inhibition of ChE's by organophosphate and
`carbamate insecticides can be explained in terms of the reaction scheme describing
`inhibition and the current model of the active site. These two concepts also provide a
`useful point of departure from which to consider less well defined but important areas
`such as the existence and properties of allosteric sites (Section 8.2).
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`Mode of action of anticholinesterases
`
`585
`
`3.1. THE REACTION SCHEME
`
`Organophosphates and carbamates react with ChE's by forming two types of
`complex. The first is a reversible complex (EI) in which the inhibitor, I, is bound to
`the enzyme, E, by non-covalent bonds. When E and I are mixed it is assumed that an
`equilibrium with Ei is quickly approached. The first part of the reaction is then
`described by the scheme,
`
`K.!
`E+I~EI
`
`SCHEMB 2.
`
`where Kd is an equilibrium dissociation constant characterizing the tightness of
`,
`binding between E and I.
`The reversible complex leads to the second complex in which part of the inhibitorls
`covalently bonded to the enzyme active-site. The covalently, bonded complex. E', is
`relatively stable, and this accounts for the 'irreversible' nature of inhibition which was
`attributed early to organophosphates. The second part of the reaction is then des(cid:173)
`cribed by the scheme.
`
`ScHEME 3.
`
`where k2 is the rate constant governing formation of the covalent enzyme-inhibitor
`complex from EI.
`Finally. E' may break down slowly' to regenerate free enzyme and products.
`Regeneration then follows the scheme.
`
`kJ
`
`E' -
`
`E + products
`
`SCHEME 4.
`
`where k3 is the regeneration rate constant.
`The overall course of inhibition is then described by combining Schemes 2, 3 and 4
`as follows:
`'
`
`K.t
`kl
`kJ
`E + I :;::::= EI ---)0 E' ---)0 E + prodUcts
`
`SCHEME 5.
`
`The power or potency of an inhibitor depends on the rate at which E' is formed and
`on its stability.
`The rate at which E' is formed depends in turn on the concentration of EI and the
`value of k2• The higher the concentration of EI and the higher k2• the faster is E'
`formed. For a given ~oncentration of E and I, the concentration of EI depends on K.t
`where
`
`~ K _ [E][I]
`fEI]
`d -
`
`(5)
`
`., ,~.-
`
`The squared brackets indicate concentrations and the concentrations are at equili(cid:173)
`brium. Thus, the smaller K d , the greater is fEI], and the faster E' will be formed.
`The inhibitory phase of the reaction then involves Schemes 2 and 3' which are
`combined below:
`
`K..
`k 2 '
`E+I~ EI---)o E'
`
`SCHEME 6.
`
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`As mentioned, the power of an inhibitor also depends on the stability of E' which is
`characterized by k3 of Scheme 4. The more stable is E' as reftected by low value of k3,
`the more potent is the inhibitor.
`If E' quickly broke down to regenerate free enzyme, the reaction would not be
`inhibit~ry no matter how fast E' were formed. Instead, Scheme 5 would characterize a
`substrate reaction, and indeed it does. Thus, by this analysis the principle difference
`between inhibition and substrate reactions of this kind are in the values of k3. For
`inhibitors k3 is small and for substrates k3 is large. Typically, k:J is millions of times
`greater for substrates than inhibitors.
`
`3.2. THE ACTIVE SITE AND THE NATURE OF THE E' COMPLEX
`
`Until the discovery that enzymes could form covalently bonded complexes with
`their inhibitors and substrates, the only"postulated enzyme-substrate or enzyme(cid:173)
`inhibitor complex was of the Michaelis-Menten type (Scheme 2) which involves only
`non-covalent bonding. Two lines of study led to the discovery of the E' c"omplex and
`its nature. One involved the use of radioactive p32-labeled organophosphates. The plZ
`was found to remain 'irreversibly' fixed to the enzyme after it had heen inhibited by
`DPp32 (Boursnell and Webb, 1949). Later, when pl2_labeled ehB was subjected to
`
`DFP
`
`(diisopropyrphosphorofluorldate)
`
`hydrolysis and amino acid analysis, the p32 was found attached to a seryl residue
`which must have been at the active site (Cohen et al., 1955: Schaffer et al., 19.54). The
`phosphate had then formed an ester by combining with the OH side group of the seryl
`residue, clearly indicating that E' was formed by a phosphorylation reaction.
`The discovery of phosphorylated seryl residues confirmed rather than initiated the
`phosphorylation theory of how organophosphates inhibited. Sometime previously
`Wilson (1951) had proposed that organophosphates inhibited by phosphorylating some
`group at the active site to form a covalently bonded phosphoryl-enzyme intermediary
`which brings us to the second line of study.
`The second line of study involved the use of hydroxylamine which was introduced
`by Hestrin (l949a) to determine the concentration of acetylchoiine (ACh). Under
`alkaline conditions hydroxylamine reacts with carboxylic esters to form hydroxamic
`acids. The reaction is analogous to that of water hydrolyzing an ester;
`
`o
`0
`RC-OR' + HOH~RC-oH + R'OH
`U
`"
`o
`0
`RC-OR' + H.NOH~RC-NOH + R'OH
`"
`U
`hydroxylamine
`hydroxamlc acid
`
`The Hestrin (l949a) method is a little unusual among the methods used to determine
`the activity of ChE's in that it measures the concentration of ACh remaining rather
`than one of the prodUcts formed. The hydrolysis of ACh proceeds far to the right
`before an eqUilibrium is reached. The concentration of acetate and choline is then
`much higher than that of ACh. However, since his method determined ACh" rather
`than its products, Hestrin was able to determine the equilibrium concentrations and to
`show that ehE catalyzed not only-""the hydrolysis, but also the synthesis of ACh until
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`Mode of action of antich·olinesterases
`an equilibrium was reached. By showing further that ChB reacted with acetate in the
`presence of hydroxylamine to form the acetylhydroxamic acid, Hestrin initiated a line
`of study which soon led Wilson and his colleagues to propose the acylation theory of
`how substrates react (e.g. Wilson, Bergmann and Nachmansohn. 1950).
`To appreciate what follows it is useful to recapitulate Hestrin's (1949b) experiments
`with acetate, hydroxylamine and ChB using the hindsight provided by Wilson's
`brilliant interpretation. Recognize first that when hydroxylamine and acetate are
`mixed at pH 7.1, nothing happens. Observe then that when ChE is added to this
`solution. acetylhydroxamic acid .begins to form. Now recall that hydroxylamine will
`react only with carboxylic esters, and further that ChB's catalyze the synthesis as well as
`the hydrolysis of ACh. The conclusion is inescapable that acetate must have combined
`with ChE to form an intermediate acetyl-ChB ester with which hydroxylamine reacted.
`We know now that the enzyme ester involves the OH of seryl, so we can summarize the
`interpretation as follows:
`
`o
`o
`0
`II
`II
`II
`-H.O
`EO-C-CHa + H.NOH .... CH3C-NOH + EOH
`EOH + HOCCH3 ~
`+H.O
`acety/ated ChE
`aeetylhydroxamlc acid
`
`where BOH is the enzyme with the seryl OH group on it. This then was the acylation
`theory of how substrates reacted with ChB's.
`It was then only a step to the phosphorylation theory. The question addressed was
`the reversibility, if any, of ChB inhibited by organophosphates which, 'in contrast to
`previously known inhibitors were con.sidered to be irreversible' (WIlson. 1951). In the
`reaction of hydroxylamine with carl10xylic esters, including the acetylated ChE, the
`relatively negative N atoms (presumably) of. hydroxylamine had made a nucleophilic
`attack. on the relatively positive carbonyl C of the ester. Hydroxylamine then
`substituted for H 20 which makes the attack in the normal deacetylation reaction
`
`o ll( -)
`II ~ carbon attacked
`.
`CH3-C-OE
`8(+)
`
`.
`
`associated with substrates. The attack by H 20 ori the comparable phosphoryl ester
`
`o
`II
`(RO)2P-O-E
`
`was ineffective in that it did not break the P-O-E bond and liberate· free enzyme.
`Wilson (1951) then substituted hydroxylamine for water. He observed that ehB
`inhibited by tetraethylpyrophosphate (TEPP) was 87.5 per cent regenerated after 5 hr
`in the presence of 1.2 M hydroxylamine, while in a control only 5 per cent of the initial
`activity had regenerated. Wilson (1951) reasoned that TEPP must have formed a
`diethylphosphoryl enzyme intermediate with ehB which hydroxylamine had attacked
`to liberate free enzyme, giving rise to the phosphorylation thepry of inhibition.
`Wilson et al. (1950) had also studied the effect of pH on the activity of ChE and had
`observed that the activity increased with pH, reaching a maximum between pH 8 and
`9 [Fig. 2(a)]. The activity then decreased as the pH increased beyond 9. The initial
`increase in activity implicated a residue with a pK of about 7.2 which would
`correspond to the imidazole group of histidine. Apparently the protonated or posi(cid:173)
`tively charged imidazole on ChE below pH 7.2 was not active. while the uncharged
`form to above pH 7.2 was active. Moreover. the imidazole nitrogens are sufficiently
`nucleophilic· to attack the esters and hydrolyze them as described above. At first it
`
`1 Q)
`0 )
`('II
`~l
`r- 0.
`.~
`t -:E
`
`c
`0
`
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`I
`EH+ ~ HT + EH ----'-'- 1£-+ H+
`.z;r---
`~I
`active
`I I nac:tive
`
`I ,
`
`6
`
`7
`
`10
`
`100
`
`110
`
`110
`
`40
`
`:-
`E :to
`~
`·SIOO.
`E ... 0 80
`
`K
`
`GO
`
`40
`
`20
`
`0
`5
`
`8
`
`7
`
`8
`pH
`FIG. 2(a). Typical pH versus activity curve. The effect of pH on the hydrolysis of acetyl(cid:173)
`choline by eel AChE is shown. (After Wilson and Bergmann, 1950.)
`
`9
`
`10
`
`11
`
`FIO.2(b). The effect of pH on spontaneous regeneratit;m of cilrbiunylated and phosphorylated
`AChE (after Aldridie and Reiner, 1m). The effect of pH on rates of inhibition of ebB's by' .
`organophosphates and carbamates are quite similar to those· shown in Figs. 2(a) and (b).
`
`was believed that one of these imidazole nitrogens was acylated during the substrate
`and inhibition reactions. The finding of a seryl phosphate, not an imidazole phosphate,
`on pll-Iabeled ChE appeared to refute the involvement of imidazole; but as Wilson
`(1960) noted, the seryl OH group is not,