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How the cholinesterases got their modern names
Chemico-Biological Interactions 187 (2010) 23–26
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
How the cholinesterases got their modern names Victor P. Whittaker a,b,∗ a b
Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Wolfson College, Cambridge, UK
a r t i c l e
i n f o
Article history: Available online 3 March 2010 Keywords: True-, pseudo-, acetyl-, butyryl-cholinesterases Carbon analogues of choline esters Enzyme–substrate complementarity Dispersion forces Ion-induced dipole interactions
a b s t r a c t The classification of the cholinesterases into ‘true’ and ‘pseudo’ became obsolete when, some 60 years ago, the author and his co-workers showed that both enzymes had a broad specificity and differed mainly in their acyl group specificity. The importance of complementarity between enzyme and substrate was shown by the high rate of hydrolysis of carbon analogues of choline esters and this enabled pioneer studies of the intermolecular forces between the enzymes’ active centres and their substrates to be carried out. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Choline-esterase is discovered and purified The instability of acetylcholine (ACh) in the presence of blood and tissues was appreciated even before it was positively identified as a transmitter [1,2]. By the early thirties of last century, this instability was recognized as being due to the presence of an enzyme which hydrolysed ACh to the physiologically inactive choline and acetate, the enzyme had been given a name, choline-esterase, later shortened to cholinesterase (ChE), and it had been partially purified from serum [3]. The ability of eserine (physostigmine) to stabilize ACh in the presence of blood and tissues was seen to be due to the drug’s ability specifically to inhibit it in low concentrations [4,5]. It thus became apparent that the pharmacological properties of eserine, and later the organophosphates, could be explained by their specific action on ChE, probably the first example of a ‘biochemical lesion’—the concept [6,7] that the consequences of vitamin deficiencies and the actions of drugs and toxins are due to interference with specific steps in metabolism rather than to a vague general effect on cells and tissues. 2. True and pseudo-cholinesterases With increasing knowledge of the specificity of ChEs of diverse origin for substrates and inhibitors came the realization that they were not all identical and attempts were made to classify them.
∗ Correspondence address: 54 Gough Way, Cambridge CB3 9LN, UK. Tel.: +44 0 1223 351577. E-mail address: [email protected]. 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.02.041
One of the first of these was made by Alles and Hawes [8] who discovered that erythrocyte ChE could hydrolyse the compound acetyl--methylcholine whereas serum ChE could not. They also noted that the rate of hydrolysis of ACh by the erythrocyte enzyme reached a maximum at a relatively low concentration, thereafter declining with increasing ACh concentration (a phenomenon known as substrate inhibition), whereas its hydrolysis by the serum enzyme conformed to normal Michaelis–Menten kinetics. Mendel et al. [9] were impressed by their finding that purified erythrocyte ChE could not hydrolyse typical aliphatic esters such as tributyrin and methyl butyrate, whereas highly purified serum ChE could. This led them to propose that ChEs of the erythrocyte type were specific or true ChEs while those of the serum-type, having aliphatic esterase activity, were non-specific or pseudo-ChEs, a terminology still occasionally used. They also found that serum-type but not erythrocyte-type ChEs would hydrolyse benzoylcholine, and proposed hydrolysis of acetyl--methylcholine and benzoylcholine as convenient criteria for classifying ChEs into the two types. The Mendel classification held up well when the inhibitors BW284C51 and isoOMPA were found to be specific for the two types of ChE. It became less logical when Bodansky [10] discovered that the erythrocyte enzyme could hydrolyse the typical aliphatic ester triacetin. This led this author and his co-workers [11–18] to make a systematic re-examination of the specificity of the two classes of ChE (reviewed by Whittaker [19,20]) based on the alternative premise that erythrocyte-type ChEs might be selective for acetates in general (acetylcholine, triacetin) and the serum-type ChEs for larger acyl groups (butyrylcholine, tributyrin, benzoylcholine), but with a narrower specificity for choline esters, exemplified by their inability to hydrolyse acetyl--methylcholine, than the so-called true ChEs.
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V.P. Whittaker / Chemico-Biological Interactions 187 (2010) 23–26
3. Acetyl and butyrylcholinesterases We showed that, in all series of esters hydrolysed, the erythrocyte-type ChE showed a marked preference for acetates and had virtually no action on butyrates, whereas the serumtype ChEs while able to hydrolyse acetates, showed a marked preference for butyrates (Fig. 1a and b). We also showed that, provided these preferences were respected, both enzymes hydrolysed a wide range of aliphatic esters. Remarkably, in the alkyl series of esters, the rate at which esters were hydrolysed was dependent on the extent to which the alkyl group approached the configuration of choline (Fig. 1c and d). Thus, in the n-alkyl series, n-butyl esters were hydrolysed fastest, but if successive carbon atoms were added, not to the end of the n-butyl chain, but in the 3-position (iso-amyl, 3,3-dimethylbutyl), the successive falls in activity seen with n-amyl, n-hexyl became a rise, with 3,3-dimethylbutyl esters (the carbon analogues of choline esters) being hydrolysed at about 60% of the rate of the corresponding choline esters. We also found that the inability of serum-type ChEs to hydrolyse -substituted choline esters was mirrored in the
aliphatic system; 1-methyl-iso-amyl (1,3-dimethyl-n-butyl) esters were not hydrolysed. Since ChEs are often accompanied by aliesterases, an important feature of our work was to use preparations of ChE freed from aliesterases by purification and to show that the specificity pattern of aliesterases with respect to aliphatic esters is quite different from that of ChEs. Additional evidence that the aliphatic esters were being hydrolysed by ChEs was provided by competition experiments and by specific inhibitors. These findings rendered the ‘true’ and ‘pseudo’ nomenclature obsolete. At a meeting of the UK Biochemical Society in May 1948 at which our initial results were presented, the well-known neurochemist Derek Richter suggested the nomenclature aceto- and butyro-cholinesterase for the two types [11,16]. We used these in our subsequent publications until they were replaced internationally by acetyl- and butyrylcholinesterase (AChE and BuChE). Many authors now prefer the abbreviation BChE and I will use it in this paper. I find it unobjectionable provided its basis in specificity is not forgotten and we do not think of the two enzymes merely as Aand B-types without reference to their specificity.
Fig. 1. The acyl and alkyl group specificity of cholinesterases towards neutral aliphatic esters. (a, b) Acyl group specificity of (a) pigeon-brain AChE (n-amyl series) (b) human serum BChE (n-amyl and n-butyl series); (c, d) alkyl group specificity of these (c, acetates; d, acetates and propionates). Note that in (a) the acetate ester is hydrolysed maximally and the butyrate negligibly by this representative AChE, whereas in (b) butyrate esters are hydrolysed maximally by this representative BChE. All other series of esters tested behaved similarly. (c, d) Alkyl group specificity of these ChEs. Note that in both (c) and (d), of the n-alkyl esters, n-butyl esters are the most rapidly hydrolysed by both ChEs, but with the alcohol moiety branched in the 3-position, the fall in the rate of hydrolysis seen with n-amyl, n-hexyl is reversed and is maximal with 3:3dimethylbutyl esters, the carbon analogues of acylcholines. These esters are hydrolysed at ca 60% of the rates of their choline analogues. Results of (a) and (c) are from [13], those of (b) and (d) from [14].
V.P. Whittaker / Chemico-Biological Interactions 187 (2010) 23–26
4. Intermediate types At least two ChEs have been reported which, while hydrolysing butyrylcholine, hydrolyse other choline esters faster. Thus Ord and Thompson [21] reported on a ChE which has an optimum for propionylcholine and the main ChE in Torpedo heart hydrolyses acetylcholine faster than butyrylcholine [22]. Since these enzymes do hydrolyse butyrylcholine, they are best classified as atypical BChEs.
5. The intermolecular forces binding substrates to cholinesterases The discovery of uncharged but rapidly hydrolyses substrates of the ChEs isosteric with choline esters enabled us to investigate the role of intermolecular forces in the activity of these enzymes. The late forties and the early fifties saw a growing interest in the structure of the active centres of the ChEs and in the nature of the intermolecular forces binding the substrate to the enzyme. It was pointed out that the substrate inhibition that uniquely occurs with AChE and its optimum substrate ACh could be quantitatively described by a modified Michaelis–Menten kinetics in which a second substrate molecule combines with the enzyme in such a way as to block the full occupation of the active centre by either molecule. Our work had shown that the specificity of the ChEs for the acyl and alkyl moieties of their substrates were independent of each other; it was thus reasonable to suppose that the second moiety of ACh attached itself to the choline (or alkyl) subsite of AChE and that this attachment might be stabilized by interaction with a negative charge (the ‘negative nitrogen-attracting group’) in this subsite. The attenuation or absence of this group in BChE would explain why substrate inhibition is not seen with this enzyme. The availability of the uncharged analogue of ACh allowed us to test this concept. By comparing ACh with its carbon analogue we could eliminate all intermolecular forces except those generated by the single positive charge on ACh. Our results [15] with AChE could indeed be modelled by postulating a single negatively charged group (e.g. an RCOO− group) in the choline subsite of the active centre, interacting with the NMe3 + group of ACh at the closest distance of approach of the two groups (ca 5 Å). With BChE the difference between the affinities of the charged and uncharged analogues was much less and could be largely accounted for by an ion-induced dipole (induction) force exerted by the charged, but not the uncharged substrate, in addition to the induced dipoleinduced dipole (dispersion or van der Waal’s) force expected from molecules in that close contact with the active centre implied by the complementarity of their structure. The presence of a single negative charge in the choline subsite of AChE was also postulated by Wilson and Bergmann [23] from a study of the effect of pH on the inhibition of the enzyme by eserine and its affinity for dimethylaminoethyl acetate (demethylated ACh). Both substrates exist in protonated (positively charged) and non-protonated (neutral) forms; the former is favoured by acid (pH 5). Eserine became more effective as an inhibitor and dimethylaminoethyl acetate more effective as a substrate when protonated, again suggesting the presence of a negative group in the choline subsite of the enzyme which they referred to as the ‘anionic site’. It is now known that the choline subsite of AChE is rich in amino acids containing aromatic rings whereas that of BChE has far fewer. Thus the postulated ion–ion interaction between ACh and AChE has been replaced by an interaction between the ACh cation and the pi-electrons of the aromatic rings. Pauling and Pressman [24] showed, by measuring the effect of changes in the polarizability of uncharged isosteric haptenic groups on their affinity for an antibody, that the main intermolecular force
25
Table 1 Log affinity ratios of haloacetates and corresponding propionates. Ester
Log (affinity ratios) Cl/Me
Br/Me
BuChE n-Butyl iso-Amyl Calculated
0.11 + 0.03 (3) 0.13 + 0.03 (3) 0.17
0.26 + 0.02 (3) 0.23 + 0.03 (3) 0.26
AChE n-Butyl iso-Amyl Calculated
0.56 + 0.07 (2) 0.59 + 0.06 (3) 0.40
0.40 + 0.06 (3) 0.32 + 0.01 (3) 0.55
Experimental values are means + range (no. of experiments). Results are from [15].
between them was the dispersion force. Such a force depends on the polarizability of the haptenic group; it declines rapidly with distance and is only effective if the haptenic group approaches the antibody molecule closely as a result of a high degree of complementarity (snugness of fit) between the haptenic group and the antibody’s binding site. Having shown that complementarity was also a defining factor in the specificity of ChEs, we asked if dispersion forces could also be the main binding force between aliphatic substrates and ChEs. Adopting Pauling’s and Pressman’s approach, we utilized as substrates n-butyl and iso-amyl propionates and their more polarizable, but almost isosteric chloro- and bromoacetates. Table 1 shows the observed and calculated affinity ratios for plasma BChE and erythrocyte AChE. For BChE, the agreement with theory was quite good, suggesting that here the dispersion force arising between the enzyme and substrates with a high degree of complementarity is, indeed, the main binding force between enzyme and substrate. With AChE, even when as here, an ion-dipole component had been factored into the calculated value, the agreement was not as good. In retrospect one has to admit that Michaelis constants are not the best measures of affinity, especially with substrates such as ACh with high turnover numbers. With the far greater knowledge we now possess of the structure of the active centre, it would be useful if this problem were to be revisited. In the nearly 60 years that have elapsed since the pioneer work described here was done, immense progress has been made: the structure of the ChEs has been elucidated; their interactions with their ligands are well understood; and the mechanism by which they catalyse the hydrolysis of their substrates has been clarified. These topics have been well covered in a recent review by Silman and Sussman [25], which inter alia, contains a message for the Xth ChE meeting in Sibenik! References [1] H.H. Dale, The action of certain esters and ethers of choline, and their relation to muscarine, J. Pharmacol. Exp. Ther. 6 (1914) 147–190. [2] O. Loewi, E. Navratil, Pflügers Arch. Ges. Physiol. Über humoralen Übertragbarkeit der Herzenwirkung, X. Über das Schicksal des Vagusstoffs 214 (1926) 678–688. [3] E. Stedman, E. Stedman, L.H. Easson, Choline-esterase. An enzyme present in the blood serum of the horse, Biochem. J. 26 (1932) 2056–2066. [4] E. Engelhart, O. Loewi, Fermentative Azetylcholinspaltung im Blut und ihre Hemmung durch Physostigmin, Naunyn-Schmiedebergs Arch. Exp. Path. Pharmakol. 150 (1930) 1–13. [5] K. Matthes, The action of blood on acetylcholine, J. Physiol. 70 (1930) 338–348. [6] R.A. Peters, The chemical lesion in vitamin B1 deficiency: application of modern biochemical analysis in its diagnosis, Lancet 227 (1936) 1161–1164. [7] R.A. Peters, Biochemical Lesions and Lethal Synthesis, Pergamon Press, Oxford, 1963. [8] G.A. Alles, R.C. Hawes, Cholinesterases in the blood of man, J. Biol. Chem. 133 (1940) 375–390. [9] B. Mendel, D.B. Mundell, H. Rudney, Studies on cholinesterase 3. Specific tests for true cholinesterase and pseudocholinesterase, Biochem. J. 37 (1943) 473–476.
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[10] O. Bodansky, Cholinesterase, Ann. N.Y. Acad. Sci. 47 (1946) 521–547. [11] D.H. Adams, V.P. Whittaker, The specificity of the human erythrocyte cholinesterase, Biochem. J. 43 (1948) xiv–xv. [12] D.H. Adams, The specificity of the human erythrocyte cholinesterase, Biochim. Biophys. Acta 3 (1949) 1–14. [13] V.P. Whittaker, The specificity of pigeon-brain cholinesterase, Biochem. J. 44 (1949) xlvi–xlvi10. [14] D.H. Adams, V.P. Whittaker, The cholinesterases of human blood I. The specificity of the plasma enzyme and its relation to the erythrocyte cholinesterase, Biochim. Biophys. Acta 3 (1949) 358–366. [15] D.H. Adams, V.P. Whittaker, The cholinesterases of human blood II. The forces acting between enzyme and substrate, Biochim. Biophys. Acta 4 (1950) 543–558. [16] L.M. Sturge, V.P. Whittaker, The esterases of horse blood 1. The specificity of horse plasma cholinesterase and aliesterase, Biochem. J. 47 (1950) 518–525. [17] L.A. Mounter, V.P. Whittaker, The esterases of horse blood 2. The specificity of horse erythrocyte cholinesterase, Biochem. J. 47 (1950) 525–530.
[18] L.A. Mounter, The specificity of cobra-venom cholinesterase, Biochem. J. 50 (1951) 122–128. [19] V.P. Whittaker, Specificity, mode of action and distribution of cholinesterases, Physiol. Rev. 31 (1951) 312–343. [20] V.P. Whittaker, The Cholinergic Neuron and its Target, Birkhäuser, Boston, 1992. [21] M. Ord, R.H.S. Thompson, The preparation of soluble cholinesterases from mammalian heart and brain, Biochem. J. 49 (1951) 191–199. [22] J.P. Toutant, J. Massoulié, S. Bon, Pseudocholinesterase in Torpedo marmorata tissues: comparative study of the catalytic and molecular properties of this enzyme with acetylcholinesterase, J. Neurochem. 44 (1985) 580–592. [23] I.B. Wilson, F. Bergmann, Acetylcholinesterase. VIII. Dissociation constants of the active groups, J. Biol. Chem. 186 (1950) 683–692. [24] L. Pauling, D. Pressman, J. Am. Chem. Soc. 67 (1945) 1003–1012. [25] I. Silman, J.L. Sussman, Acetylcholinesterase: how is its structure related to function? Chem.-Biol. Interact. 175 (2008) 3–10.
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
How the cholinesterases got their modern names Victor P. Whittaker a,b,∗ a b
Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Wolfson College, Cambridge, UK
a r t i c l e
i n f o
Article history: Available online 3 March 2010 Keywords: True-, pseudo-, acetyl-, butyryl-cholinesterases Carbon analogues of choline esters Enzyme–substrate complementarity Dispersion forces Ion-induced dipole interactions
a b s t r a c t The classification of the cholinesterases into ‘true’ and ‘pseudo’ became obsolete when, some 60 years ago, the author and his co-workers showed that both enzymes had a broad specificity and differed mainly in their acyl group specificity. The importance of complementarity between enzyme and substrate was shown by the high rate of hydrolysis of carbon analogues of choline esters and this enabled pioneer studies of the intermolecular forces between the enzymes’ active centres and their substrates to be carried out. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Choline-esterase is discovered and purified The instability of acetylcholine (ACh) in the presence of blood and tissues was appreciated even before it was positively identified as a transmitter [1,2]. By the early thirties of last century, this instability was recognized as being due to the presence of an enzyme which hydrolysed ACh to the physiologically inactive choline and acetate, the enzyme had been given a name, choline-esterase, later shortened to cholinesterase (ChE), and it had been partially purified from serum [3]. The ability of eserine (physostigmine) to stabilize ACh in the presence of blood and tissues was seen to be due to the drug’s ability specifically to inhibit it in low concentrations [4,5]. It thus became apparent that the pharmacological properties of eserine, and later the organophosphates, could be explained by their specific action on ChE, probably the first example of a ‘biochemical lesion’—the concept [6,7] that the consequences of vitamin deficiencies and the actions of drugs and toxins are due to interference with specific steps in metabolism rather than to a vague general effect on cells and tissues. 2. True and pseudo-cholinesterases With increasing knowledge of the specificity of ChEs of diverse origin for substrates and inhibitors came the realization that they were not all identical and attempts were made to classify them.
∗ Correspondence address: 54 Gough Way, Cambridge CB3 9LN, UK. Tel.: +44 0 1223 351577. E-mail address: [email protected]. 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.02.041
One of the first of these was made by Alles and Hawes [8] who discovered that erythrocyte ChE could hydrolyse the compound acetyl--methylcholine whereas serum ChE could not. They also noted that the rate of hydrolysis of ACh by the erythrocyte enzyme reached a maximum at a relatively low concentration, thereafter declining with increasing ACh concentration (a phenomenon known as substrate inhibition), whereas its hydrolysis by the serum enzyme conformed to normal Michaelis–Menten kinetics. Mendel et al. [9] were impressed by their finding that purified erythrocyte ChE could not hydrolyse typical aliphatic esters such as tributyrin and methyl butyrate, whereas highly purified serum ChE could. This led them to propose that ChEs of the erythrocyte type were specific or true ChEs while those of the serum-type, having aliphatic esterase activity, were non-specific or pseudo-ChEs, a terminology still occasionally used. They also found that serum-type but not erythrocyte-type ChEs would hydrolyse benzoylcholine, and proposed hydrolysis of acetyl--methylcholine and benzoylcholine as convenient criteria for classifying ChEs into the two types. The Mendel classification held up well when the inhibitors BW284C51 and isoOMPA were found to be specific for the two types of ChE. It became less logical when Bodansky [10] discovered that the erythrocyte enzyme could hydrolyse the typical aliphatic ester triacetin. This led this author and his co-workers [11–18] to make a systematic re-examination of the specificity of the two classes of ChE (reviewed by Whittaker [19,20]) based on the alternative premise that erythrocyte-type ChEs might be selective for acetates in general (acetylcholine, triacetin) and the serum-type ChEs for larger acyl groups (butyrylcholine, tributyrin, benzoylcholine), but with a narrower specificity for choline esters, exemplified by their inability to hydrolyse acetyl--methylcholine, than the so-called true ChEs.
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V.P. Whittaker / Chemico-Biological Interactions 187 (2010) 23–26
3. Acetyl and butyrylcholinesterases We showed that, in all series of esters hydrolysed, the erythrocyte-type ChE showed a marked preference for acetates and had virtually no action on butyrates, whereas the serumtype ChEs while able to hydrolyse acetates, showed a marked preference for butyrates (Fig. 1a and b). We also showed that, provided these preferences were respected, both enzymes hydrolysed a wide range of aliphatic esters. Remarkably, in the alkyl series of esters, the rate at which esters were hydrolysed was dependent on the extent to which the alkyl group approached the configuration of choline (Fig. 1c and d). Thus, in the n-alkyl series, n-butyl esters were hydrolysed fastest, but if successive carbon atoms were added, not to the end of the n-butyl chain, but in the 3-position (iso-amyl, 3,3-dimethylbutyl), the successive falls in activity seen with n-amyl, n-hexyl became a rise, with 3,3-dimethylbutyl esters (the carbon analogues of choline esters) being hydrolysed at about 60% of the rate of the corresponding choline esters. We also found that the inability of serum-type ChEs to hydrolyse -substituted choline esters was mirrored in the
aliphatic system; 1-methyl-iso-amyl (1,3-dimethyl-n-butyl) esters were not hydrolysed. Since ChEs are often accompanied by aliesterases, an important feature of our work was to use preparations of ChE freed from aliesterases by purification and to show that the specificity pattern of aliesterases with respect to aliphatic esters is quite different from that of ChEs. Additional evidence that the aliphatic esters were being hydrolysed by ChEs was provided by competition experiments and by specific inhibitors. These findings rendered the ‘true’ and ‘pseudo’ nomenclature obsolete. At a meeting of the UK Biochemical Society in May 1948 at which our initial results were presented, the well-known neurochemist Derek Richter suggested the nomenclature aceto- and butyro-cholinesterase for the two types [11,16]. We used these in our subsequent publications until they were replaced internationally by acetyl- and butyrylcholinesterase (AChE and BuChE). Many authors now prefer the abbreviation BChE and I will use it in this paper. I find it unobjectionable provided its basis in specificity is not forgotten and we do not think of the two enzymes merely as Aand B-types without reference to their specificity.
Fig. 1. The acyl and alkyl group specificity of cholinesterases towards neutral aliphatic esters. (a, b) Acyl group specificity of (a) pigeon-brain AChE (n-amyl series) (b) human serum BChE (n-amyl and n-butyl series); (c, d) alkyl group specificity of these (c, acetates; d, acetates and propionates). Note that in (a) the acetate ester is hydrolysed maximally and the butyrate negligibly by this representative AChE, whereas in (b) butyrate esters are hydrolysed maximally by this representative BChE. All other series of esters tested behaved similarly. (c, d) Alkyl group specificity of these ChEs. Note that in both (c) and (d), of the n-alkyl esters, n-butyl esters are the most rapidly hydrolysed by both ChEs, but with the alcohol moiety branched in the 3-position, the fall in the rate of hydrolysis seen with n-amyl, n-hexyl is reversed and is maximal with 3:3dimethylbutyl esters, the carbon analogues of acylcholines. These esters are hydrolysed at ca 60% of the rates of their choline analogues. Results of (a) and (c) are from [13], those of (b) and (d) from [14].
V.P. Whittaker / Chemico-Biological Interactions 187 (2010) 23–26
4. Intermediate types At least two ChEs have been reported which, while hydrolysing butyrylcholine, hydrolyse other choline esters faster. Thus Ord and Thompson [21] reported on a ChE which has an optimum for propionylcholine and the main ChE in Torpedo heart hydrolyses acetylcholine faster than butyrylcholine [22]. Since these enzymes do hydrolyse butyrylcholine, they are best classified as atypical BChEs.
5. The intermolecular forces binding substrates to cholinesterases The discovery of uncharged but rapidly hydrolyses substrates of the ChEs isosteric with choline esters enabled us to investigate the role of intermolecular forces in the activity of these enzymes. The late forties and the early fifties saw a growing interest in the structure of the active centres of the ChEs and in the nature of the intermolecular forces binding the substrate to the enzyme. It was pointed out that the substrate inhibition that uniquely occurs with AChE and its optimum substrate ACh could be quantitatively described by a modified Michaelis–Menten kinetics in which a second substrate molecule combines with the enzyme in such a way as to block the full occupation of the active centre by either molecule. Our work had shown that the specificity of the ChEs for the acyl and alkyl moieties of their substrates were independent of each other; it was thus reasonable to suppose that the second moiety of ACh attached itself to the choline (or alkyl) subsite of AChE and that this attachment might be stabilized by interaction with a negative charge (the ‘negative nitrogen-attracting group’) in this subsite. The attenuation or absence of this group in BChE would explain why substrate inhibition is not seen with this enzyme. The availability of the uncharged analogue of ACh allowed us to test this concept. By comparing ACh with its carbon analogue we could eliminate all intermolecular forces except those generated by the single positive charge on ACh. Our results [15] with AChE could indeed be modelled by postulating a single negatively charged group (e.g. an RCOO− group) in the choline subsite of the active centre, interacting with the NMe3 + group of ACh at the closest distance of approach of the two groups (ca 5 Å). With BChE the difference between the affinities of the charged and uncharged analogues was much less and could be largely accounted for by an ion-induced dipole (induction) force exerted by the charged, but not the uncharged substrate, in addition to the induced dipoleinduced dipole (dispersion or van der Waal’s) force expected from molecules in that close contact with the active centre implied by the complementarity of their structure. The presence of a single negative charge in the choline subsite of AChE was also postulated by Wilson and Bergmann [23] from a study of the effect of pH on the inhibition of the enzyme by eserine and its affinity for dimethylaminoethyl acetate (demethylated ACh). Both substrates exist in protonated (positively charged) and non-protonated (neutral) forms; the former is favoured by acid (pH 5). Eserine became more effective as an inhibitor and dimethylaminoethyl acetate more effective as a substrate when protonated, again suggesting the presence of a negative group in the choline subsite of the enzyme which they referred to as the ‘anionic site’. It is now known that the choline subsite of AChE is rich in amino acids containing aromatic rings whereas that of BChE has far fewer. Thus the postulated ion–ion interaction between ACh and AChE has been replaced by an interaction between the ACh cation and the pi-electrons of the aromatic rings. Pauling and Pressman [24] showed, by measuring the effect of changes in the polarizability of uncharged isosteric haptenic groups on their affinity for an antibody, that the main intermolecular force
25
Table 1 Log affinity ratios of haloacetates and corresponding propionates. Ester
Log (affinity ratios) Cl/Me
Br/Me
BuChE n-Butyl iso-Amyl Calculated
0.11 + 0.03 (3) 0.13 + 0.03 (3) 0.17
0.26 + 0.02 (3) 0.23 + 0.03 (3) 0.26
AChE n-Butyl iso-Amyl Calculated
0.56 + 0.07 (2) 0.59 + 0.06 (3) 0.40
0.40 + 0.06 (3) 0.32 + 0.01 (3) 0.55
Experimental values are means + range (no. of experiments). Results are from [15].
between them was the dispersion force. Such a force depends on the polarizability of the haptenic group; it declines rapidly with distance and is only effective if the haptenic group approaches the antibody molecule closely as a result of a high degree of complementarity (snugness of fit) between the haptenic group and the antibody’s binding site. Having shown that complementarity was also a defining factor in the specificity of ChEs, we asked if dispersion forces could also be the main binding force between aliphatic substrates and ChEs. Adopting Pauling’s and Pressman’s approach, we utilized as substrates n-butyl and iso-amyl propionates and their more polarizable, but almost isosteric chloro- and bromoacetates. Table 1 shows the observed and calculated affinity ratios for plasma BChE and erythrocyte AChE. For BChE, the agreement with theory was quite good, suggesting that here the dispersion force arising between the enzyme and substrates with a high degree of complementarity is, indeed, the main binding force between enzyme and substrate. With AChE, even when as here, an ion-dipole component had been factored into the calculated value, the agreement was not as good. In retrospect one has to admit that Michaelis constants are not the best measures of affinity, especially with substrates such as ACh with high turnover numbers. With the far greater knowledge we now possess of the structure of the active centre, it would be useful if this problem were to be revisited. In the nearly 60 years that have elapsed since the pioneer work described here was done, immense progress has been made: the structure of the ChEs has been elucidated; their interactions with their ligands are well understood; and the mechanism by which they catalyse the hydrolysis of their substrates has been clarified. These topics have been well covered in a recent review by Silman and Sussman [25], which inter alia, contains a message for the Xth ChE meeting in Sibenik! References [1] H.H. Dale, The action of certain esters and ethers of choline, and their relation to muscarine, J. Pharmacol. Exp. Ther. 6 (1914) 147–190. [2] O. Loewi, E. Navratil, Pflügers Arch. Ges. Physiol. Über humoralen Übertragbarkeit der Herzenwirkung, X. Über das Schicksal des Vagusstoffs 214 (1926) 678–688. [3] E. Stedman, E. Stedman, L.H. Easson, Choline-esterase. An enzyme present in the blood serum of the horse, Biochem. J. 26 (1932) 2056–2066. [4] E. Engelhart, O. Loewi, Fermentative Azetylcholinspaltung im Blut und ihre Hemmung durch Physostigmin, Naunyn-Schmiedebergs Arch. Exp. Path. Pharmakol. 150 (1930) 1–13. [5] K. Matthes, The action of blood on acetylcholine, J. Physiol. 70 (1930) 338–348. [6] R.A. Peters, The chemical lesion in vitamin B1 deficiency: application of modern biochemical analysis in its diagnosis, Lancet 227 (1936) 1161–1164. [7] R.A. Peters, Biochemical Lesions and Lethal Synthesis, Pergamon Press, Oxford, 1963. [8] G.A. Alles, R.C. Hawes, Cholinesterases in the blood of man, J. Biol. Chem. 133 (1940) 375–390. [9] B. Mendel, D.B. Mundell, H. Rudney, Studies on cholinesterase 3. Specific tests for true cholinesterase and pseudocholinesterase, Biochem. J. 37 (1943) 473–476.
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[10] O. Bodansky, Cholinesterase, Ann. N.Y. Acad. Sci. 47 (1946) 521–547. [11] D.H. Adams, V.P. Whittaker, The specificity of the human erythrocyte cholinesterase, Biochem. J. 43 (1948) xiv–xv. [12] D.H. Adams, The specificity of the human erythrocyte cholinesterase, Biochim. Biophys. Acta 3 (1949) 1–14. [13] V.P. Whittaker, The specificity of pigeon-brain cholinesterase, Biochem. J. 44 (1949) xlvi–xlvi10. [14] D.H. Adams, V.P. Whittaker, The cholinesterases of human blood I. The specificity of the plasma enzyme and its relation to the erythrocyte cholinesterase, Biochim. Biophys. Acta 3 (1949) 358–366. [15] D.H. Adams, V.P. Whittaker, The cholinesterases of human blood II. The forces acting between enzyme and substrate, Biochim. Biophys. Acta 4 (1950) 543–558. [16] L.M. Sturge, V.P. Whittaker, The esterases of horse blood 1. The specificity of horse plasma cholinesterase and aliesterase, Biochem. J. 47 (1950) 518–525. [17] L.A. Mounter, V.P. Whittaker, The esterases of horse blood 2. The specificity of horse erythrocyte cholinesterase, Biochem. J. 47 (1950) 525–530.
[18] L.A. Mounter, The specificity of cobra-venom cholinesterase, Biochem. J. 50 (1951) 122–128. [19] V.P. Whittaker, Specificity, mode of action and distribution of cholinesterases, Physiol. Rev. 31 (1951) 312–343. [20] V.P. Whittaker, The Cholinergic Neuron and its Target, Birkhäuser, Boston, 1992. [21] M. Ord, R.H.S. Thompson, The preparation of soluble cholinesterases from mammalian heart and brain, Biochem. J. 49 (1951) 191–199. [22] J.P. Toutant, J. Massoulié, S. Bon, Pseudocholinesterase in Torpedo marmorata tissues: comparative study of the catalytic and molecular properties of this enzyme with acetylcholinesterase, J. Neurochem. 44 (1985) 580–592. [23] I.B. Wilson, F. Bergmann, Acetylcholinesterase. VIII. Dissociation constants of the active groups, J. Biol. Chem. 186 (1950) 683–692. [24] L. Pauling, D. Pressman, J. Am. Chem. Soc. 67 (1945) 1003–1012. [25] I. Silman, J.L. Sussman, Acetylcholinesterase: how is its structure related to function? Chem.-Biol. Interact. 175 (2008) 3–10.