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Home , Carboxylesterase, Cholinesterase, Esterase, Hydrolase

SESSION It Bull. Org. mond. Sante Bull. Wid Hith Org. 11971, 44, 81-89

Comparative Aspects of the Purification and Properties of Cholinesterases

KLAS-BERTIL AUGUSTINSSON I

Recent years have seen great progress in the purification and characterization of cholinesterases. Investigation has indicated the existence of two principal groups: a fairly homogeneous group of and a group of that utilize butyryl- , propionycholine, or benzoylcholine as substrates and that differ widely in their properties. This paper reviews the different types ofcholinesterase and their sources, the importance of a proper choice of in studies, methods for the purification of cholinesterases, and some of the properties of these enzymes.

Cholinesterases (ChE) constitute a group of - cholinesterases. Detailed references will be found in ases that hydrolyse choline at a higher rate recent review articles and monographs (Augustinsson, than other esters, provided the rates 1948, 1961, 1963, 1971; Gerebtzoff, 1959; Goedde are compared at optimum and controlled conditions. et al., 1967; Pilz, 1970; Usdin, 1970; Wilkinson, All cholinesterases, with a few exceptions among 1970). lower vertebrates and invertebrates, are inhibited by 1OtM . This inhibitory property and TYPES OF CHOLINESTERASE AND THEIR SOURCES the substrate specificity distinguish cholinesterases from (3.1.1. 1.), although both types Various types of cholinesterase can be differen- of are sensitive to organophosphorus com- tiated by the use of either specific substrates or selec- pounds. There seems to be a close relation between tive inhibitors and by studying kinetic behaviour. the two types, and it has been suggested that they Such investigations have indicated the existence of have a common phylogenetic origin (Augustinsson, two main groups of choline-ester-splitting enzymes, 1968). The reaction mechanism for the two enzymes the general properties of which are summarized in (cholinesterases and carboxylesterases) is similar: Table 1. the esterase reacts with the ester to produce an inter- Acetylcholinesterases (AChE) mediary acyl- complex, which can react with a variety of acyl acceptors, including water. The The best known of the choline-ester-hydrolysing inhibition of these by certain phosphoryl, enzymes are the acetylcholinesterases (3.1.1.7), which carbamyl, and sulfonyl derivatives can be explained use as their natural substrate. They by an analogous mechanism. appear to be integral parts of certain electrogenic Esterases resistant to both physostigmine and or- membranes and other isoluble structures. The gano- compounds are represented by the main sources are brain and nervous tissues, erythro- arylesterases (3.1.1.2), which hydrolyse aromatic cytes, and electric organs. An enzyme with similar esters at particularly high rates, and the acetyl- properties is present in cobra venom, where it seems esterases (3.1.1.6), which act preferentially on acetic- to be in solution. Specificity and kinetic behaviour acid esters. are quite similar for most of these esterases, and the This paper briefly examines the general compara- widely divergent properties characterizing other types tive aspects of the properties and purification of of cholinesterase from various sources are generally not observed. in 1 Associate Professor of , Biochemical Insti- AChE is sometimes present other tissues and tute, University of Stockholm, Sweden. organs together with other types of cholinesterase.

2616 -81- 82 K.-B. AUGUSTINSSON

Table 1 Some general properties of cholinesterases

Property Acetylcholinesterases a | Cholinesterases b

source electric organ of the electric serum, pancreas, heart, eel, brain, erythrocytes, cobra venom optimum substrate acetylcholine , propionylcho- line, or benzoylcholine utilization of acetyl-,-methylcholine substrate non-substrate species differences not significant significant inhibition by: quaternary ammonium compounds +++ + bis-N,N'-diisopropylphosphoro- + + + + diamidic anhydride c phenothiazine derivatives + ++

a Systematic name: acetylcholine acetyl- (3.1.1.7). b Systematic name: acylcholine acyl-hydrolase (3.1.1.8). c The so-called - iso-OMPA".

Plasma cholinesterases erties of both a and a cholinesterase. Cholinesterases (3.1.1.8) different from those dis- It is the only known example of an physostigmine- cussed above are present in the plasma of sensitive esterase that hydrolyses non-choline esters humans and higher vertebrates. With reference to more rapidly than choline esters. This enzyme is re- substrate specificity, one can distinguish between garded as an intermediate stage in the phylogenetic butyrylcholinesterases (BuChE), propionylcholin- evolution of plasma esterases. Mutational changes esterases (PrChE), and benzoylcholinesterases are likely to have led to the development of esterases (BzChE). However, such terms carry no implica- with highly divergent substrate specificities. It has tions as to the physiological substrates for these been suggested that BuChE is one of the last en- enzymes, which are still unknown. These enzymes zymes to arise as the result of these mutational are also present in various organs-e.g., liver, pan- changes during the phylogenetic evolution. creas, intestine, heart, and muscle. Several of these The cholinesterases from a variety of fowl have sources contain mixtures of various types of cholin- unusual properties. For example, the cholinesterase esterase together with both carboxylesterases and of avian plasma has most of the properties of a arylesterases (Holmes et al., 1968). PrChE, but it can hydrolyse acetyl-,B-methylcholine, There are marked differences in cholinesterases which is usually considered to be a substrate only from different species (Augustinsson, 1968). The for AChE. Generally, in lower vertebrates, PrChE plasma of the rat, the rabbit, the cock, and probably are much more abundant than are BuChE. The the frog are characterized by PrChE, the properties plasma of teleostian fish and elasmobranchs contain of which are different in each species with regard an esterase that can be designated an AChE on the to inhibitor-sensitivity and kinetic behaviour. Thc basis of its subsLrate specificity but that differs from BuChE in the plasma of man, the horse, and the AChE in kinetic behaviour (Augustinsson, 1959, dog show similar properties but have different 1968). molecular forms as shown by electrophoresis. Cholin- Substrate inhibition is one of the characteristics esterases also exist in multiple forms, as variants of that distinguish AChE from other types of cholin- normal types, and as isoenzymes. esterase. However, an enzyme that has been isolated from the muscle of the plaice (Pleuronectes platessa) Other cholinesterases is inhibited by excess substrate, but its optimum The turtle plasma esterase has several unique substrate is butyrylcholine (Lundin, 1967). characteristics (Augustinsson, 1968), exhibiting prop- preparations have been found to exhibit a cholin- PURIFICATION AND PROPERTIES OF CHOLINESTERASES 83 esterase activity that is regarded as a special type SPECIFICITY AND CHOICE OF SUBSTRATE of esterase. IN CHOLINESTERASE STUDIES Plants and other lower organisms have no cholin- Since cholinesterases constitute a group of ester- esterase activity, and in all probability this is also ases with widely divergent properties, it is advisable true of bacteria. An AChE-like enzyme was induced to state the source of the enzyme used in any work by treating Pseudomonasfluorescens with choline and with these enzymes. It is not possible to extrapolate its derivatives. This enzyme has been purified and results obtained with the plasma or an organ of one found to be resistant to organophosphorus com- species to those of any other species. Intermediate pounds. Its does not contain serine, but types between " specific" cholinesterases, such as has instead an -binding group (Fitch, 1964). BuChE, PrChE, and AChE, also exist. The presence of cholinesterases in protozoans, It is generally accepted that no cholinesterase so sponges, and hydrozoans has been reported but the far investigated has an absolute specificity for choline results seem to be highly dependent on the sensitivity esters. In fact, all cholinesterases also split ordinary of the methods used. The activity is usually extreme- esters, the various enzymes having distinct specificity ly low, but since insufficient biochemical information patterns. Thus, for example, AChE splits acetic is available, the type(s) of enzyme present cannot acid esters more rapidly than propionic or butyric be determined. acid esters, whereas human plasma BuChE catalyses Commercial cholinesterases the hydrolysis of esters at a higher rate than the esters of the lower homologous acids. As AChE is available from two sources, the electric mentioned above, there are other types that split organ of the electric eel (Electrophorus electricus) propionic acid esters at the highest rate. This rule and bovine erythrocytes. Plasma BuChE is available is valid for choline as well as for non-choline esters. from the plasma of either man or the horse. Some Consequently, any acetate should be a more favour- properties of these cholinesterases are given in able substrate for AChE than for other cholineste- Table 2. The molecular activity is given in terms of rases, and a butyrate can be expected to be a useful ,umoles of acetylcholine hydrolysed per mole of substrate for human serum BuChE. active centre per minute. Solution molarities were There are, however, a great many in determined (Usdin, 1970) by measuring the hydrolysis a crude enzyme preparation, which can be responsible rate at appropriate enzyme concentrations and under for the reaction when using a non-choline ester as optimum experimental conditions, calculating the substrate. The main problem in the use of non- rate that would be expected if the enzyme concentra- choline esters in such studies, therefore, is to deter- tion were 1 mg/ml, and then determining the molar- mine whether the ester in question is actually split ity using reported values for molecular activity. by a cholinesterase alone or by another esterase as well. The generalization may be made that any ester Table 2 can be used as a substrate for assaying ChE activity if the preparation studied contains only ChE and Commercial cholinesterases * if the specificity of the esterase activity is known in detail. For example, a preparation containing AChE Molecular Molarity (ILM) Type and source activitya of solution as the only esterase can be studied with any ester (M4moles)(,umoles) containing1.0 mg/ml split by this enzyme. As long as choline esters with more or less selective specificity for various cholinesterases are available, such esters are preferable to the less specific non- electric organ 720 000 25 choline esters. In special cases, however, certain bovine erythrocytes 295 000 9.4 x 10-2 non-choline esters may be of great value-for exam- ple, in the histochemical detection of these enzymes human plasma 50 000 b 2.2 x 1 0-1 and in the detection of ChE in chromatograms or electropherograms. Particularly useful equine serum 50 000 9.4 x 1 substrates are 0-2 those that, on hydrolysis, give reaction products that have characteristic colours or that are easily detected 'Adapted from Usdin (1 970). a See text. by spectrophotometric, fluorometric, or radiometric bi Estimated value. techniques. 84 K.-B. AUGUSTINSSON

Some of the most frequently used substrates for organ of the electric eel (Electrophorus electricus). esterase determinations are 1-naphthyl acetate and The purification procedures have been developed to certain other esters of 1-naphthol or 2-naphthol. a large extent in the laboratory of David Nachman- The isoenzyme status of esterases in vertebrate tis- sohn at Columbia University, New York. Most sues was based on the use of 1-naphthyl acetate and procedures start with mucin-free preparations, which some related 1- and 2-naphthyl derivatives. The pro- are subjected to ammonium sulfate fractionation at cedure using 1-naphthyl acetate can be used for the various pH values, chromatography on ion-exchange quantitative determination of esterase activities after celluloses, and gel filtration (Sephadex G-200) (Leu- starch gel electrophoresis. It should be remembered zinger & Baker, 1967). Leuzinger et al. (1969) have that the use of a naphthyl ester as a substrate for succeeded in purifying this enzyme to the state of esterase activity will not give a picture of all esterases crystallinity and have, as the result of careful present, because some may not split this type of chemical and X-ray crystallographic analysis, de- ester. Furthermore, 1-naphthyl acetate, for example, scribed the crystals in detail. The best preparation is a nonspecific substrate for several forms of ester- obtained was purified 720 times and had a specific ase, for which it has different affinities. However, in activity of 750 000 (,umoles of acetylcholine hydro- comparative studies of species and of tissues, the lysed per hour per mg of protein). relative specific activities towards 1-naphthyl acetate Solubility studies with and without (Mas- or a similar substrate can be a useful measure, pro- soulie et al., 1970) demonstrated the occurrence of vided it is stated what activities are measured and three different molecular species of AChE, differing what types of esterase are not detected. in their sedimentation coefficients. A fourth species was yielded after trypsin treatment and is similar PURIFICATION OF CHOLINESTERASES to purified AChE (see also below, under Molecular weight). Some major problems involved in the purification of cholinesterases are the insolubility of AChE from Erythrocytes. Various methods for purifying AChE most sources, the sensitivity of plasma cholinesterases from erythrocytes have been reported. They differ to denaturation by organic solvents, and the high principally in the techniques used for dissolving the molecular weights of most of these enzymes. Several enzyme out of the stroma. Red cells of different of the cholinesterases are activated rather than animals vary in the ease with which enzymes are inactivated by organic solvents. rendered soluble; for example, the enzyme is more Various techniques have been recommended for tightly bound to the membrane of human cells than rendering AChE soluble as the first stage in purifica- to that of bovine cells (Mitchell & Hanahan, 1966). tion, such as treatment with surface-active agents The enzyme has been brought into solution with (e.g., taurocholate) or with lipolytic or proteolytic butanol, ammonia, chloroform, lysolecithin, Tween enzymes (e.g., trypsin and pancreatopeptidase). 20, and Triton X-I 00 in 8M urea; it has also been Highly stable solutions of brain AChE were obtained rendered soluble by ultrasonic vibration. when the homogenate was first digested with pan- creatopeptidase and then frozen, thawed, and treated Brain. As with erythrocytes, AChE from brain with sulfate (Kaplay & Jagannathan, must be rendered soluble before any purification 1970). steps can be taken. The isolated subcellular particles Numerous methods for purifying cholinesterases (mitochondria and microsomes) are treated in vari- have been reported. Some of these methods and ous ways (see above), the most successful procedure some of the properties of the highest-purity prepara- being the use of pancreatopeptidase. This method tions yet obtained are listed in Table 3. Information was recently used by Kaplay & Jagannathan (1970) on methods of purification and on the properties with caudate nucleus of ox brain, and yielded a of purified enzyme preparations can be found in highly stable, soluble enzyme purified 5 000-fold. recent reviews (Augustinsson, 1963; Witter, 1963; Usdin, 1970). Muscle. Cholinesterases are also firmly bound to skeletal muscle, a tissue that has been used as an Acetylcholinesterases enzyme source by a group of Hungarian workers Electric organ. The most active and purest AChE (K6ver et al., 1964). A highly active preparation obtained so far is that prepared from the electric was obtained from rabbit muscle (specific activity PURIFICATION ANt) PROPERTIES OF CHOLINESTERASES 85

Table 3 Purest cholinesterase preparations obtained from different sources 1- Approx. Type and source degree Specific Molecular Molecular Refere cesd of cholinesterase Method of purification of purification activity (l weight b activity C (x-fold) acetylcholinesterase electric organ of ammonium sulfate fractionation 720 750 000 260 000 740 000 5, 6 electric eel at different pH values; cellulose ion-exchange cellulose-phosphate and DEAE-Sephadex chromato- graphy ox erythrocytes butanol extraction; ammonium 250-400 250 300 000 1, 8 sulfate fractionation ox brain digestion with pancreatopeptidase 5 000 100 000 160 000- c-.-430 000 4 (3.4.4.7); freezing and thawing; 360 000 e treatment with ; ammonium sulfate fractionation; DEAE-cellulose, calcium-phos- phate, and Sephadex G-200 chro- matography

head of housefly butanol extraction; ammonium 157 1 630 3 000 000- 100 000 2 sulfate fractionation; calcium phos- 4 000 000 phate fractionation; acetonie frac- tionation 8 biityrylcholinesterase human serum ammonium sulfate fractionation; 10 000 3 180 348 000 84 000 3 chromatography with alumina and with Sephadex G-200 porcine parotid ammonium sulfate fractionation; 3 220 74 000 368 000 9 gland chromatography with DEAE-Se- phadex, with carboxymethylccllu- lose, and with Sephadex G-200 body muscle of plaice autolysis with bacteria; ammo- 2 000 3180 nium sulfate fractionation; Sephadex G-200 chromatography

a pLMoles of acetylcholine hydrolysed per hour per mg of protein. The values listed should be accepted only with caution, since the activity was measured under different experimental conditions (e.g., substrate concentration, pH, and temperature) and by assay methods that are not comparable. b Determined by various techniques, mostly by sedimentation diffusion. The values depend on the method used. c Moles of acetylcholine hydrolysed per mole of active centre per minute (also known as the " turnover number"). d References: 1, Cohen & Warringa (1 953); 2, Dauterman et al. (1 962); 3, Haupt et al. (1 966); 4, Kaplay & Jagannathan (1 970); 5, Leuzinger & Baker (1967); 6, Leuzinger et al. (1969); 7, Lundin (1967); 8, Mitchell & Hanahan (1966); 9, Tucci & Seifter (1969) e Determined with a similar preparation (Jackson & Aprison, 1 966). For a discussion of the molecular size and multiplicity of cholinester- ases in , see Krysan & Kruckeberg (1970).

390 000), and several myosin preparationis were Bultyrylcholiniesterases found to be active. Blood plasmna. Several successful attempts have A special procedure for purifying a cholinesterase, been made to purify BuChE from human and horse best regarded as a BuChE, from the body muscle plasma or serum, the sources in which the highest of the plaice (Pleiurontectes platessa) was described activity of this enzyme is found. The most frequently by Lundin (1967). The enzyme was liberated by used method is ammonium sulfate fractionation incubating muscle homogenate with bacteria (Cyto- followed by preparative electrophoresis (Heilbronn, phaga sp.); it could then be purified about 2 000-fold 1962), or by ultracentrifugation preceding the electro- to give a preparation of relatively high specific phoresis (Jansz & Cohen, 1962). By means of the activity. latter technique a 14000-fold purification of horse 86 K.-B. AUGUSTINSSON serum BuChE was achieved. A homogeneous prepa- to ion-exchangers, Sepharose, or other particulate ration, purified 10 000-fold, was obtained from matter. Such enzyme preparations generally have human serum using adsorption to aluminium hydrox- enhanced stability during storage. ide, zone electrophoresis, and gel filtration on Sephadex G-200 (Haupt et al., 1966). This prepara- SOME PROPERTIES OF CHOLINESTERASES tion was further purified by selective elution with choline from a DEAE-cellulose column (Yoshida, Molecular weight 1970). The molecular weight of the purest BuChE Different values have been reported for the mole- has been calculated to be about 300 000 (Svensmark, cular weight of purified AChE (see Table 3). Ultra- 1965; cf. Table 3). centrifugation of AChE isolated from the electric Parotid gland. A most successful preparation of organ has yielded either 3 sedimentation peaks or BuChE from porcine parotid glands, which are a a single peak with a sedimentation coefficient of rich source of this enzyme, was recently achieved 10.8 S, corresponding to a molecular weight of by Tucci & Seifter (1969) using the same general 230 000. The molecular weight of polymeric material techniques as described for other cholinesterases. has been determined by various methods (diffusion The over-all purification was more than 3 000-fold and light scattering) to be about 30 000 000. and the specific activity of the final was The molecular size seems to be dependent on the 74000. The purified enzyme behaved as a single pH and ionic strength of the medium used in isolat- component (a monodisperse system) in sedimenta- ing the AChE. This enzyme is polydispersed in tion velocity and sedimentation equilibrium centri- media of low ionic strength, but exhibits only one fugation, and migrated as a single esterase in electro- sedimentation coefficient (14 S) in solutions of higher phoresis with an estimated molecular weight of ionic strength. The moiety sedimenting at 4 S is 368 000. probably the monomer of AChE, that sedimenting at 8-10 S the dimer, and that sedimenting at 12-14 S Other cholinesterases the trimer. Brain AChE has been separated into four moieties Cholinesterases with various properties, both simi- on a Sephadex G-200 column. It should be remem- lar and dissimilar to AChE and BuChE, have been bered that one or more of such enzyme forms might purified from a number of sources-e.g., cobra be the result of the procedure used to bring the venom, pancreas, liver, blood, and the pancreatic enzyme into solution. The 4 isoenzymes of AChE juice of Helix pomatia. The inducible cholinesterase previously identified in housefly brain have molecular of Pseudomonas fiuorescens (Goldstein strain) was weights of less than 500 000 (Eldefrawi et al., 1970). recently purified by carboxymethylcellulose-Sepha- The molecular weight of purified horse or human dex chromatography to give a final preparation serum BuChE has been shown to exceed 200 000. with a specific activity of about 2 000. None of Ultracentrifugation studies gave a value of approxi- these preparations had particularly high purity. mately 300 000. A value of 750 000 for horse These and similar preparations are of interest pri- serum BuChE was obtained from inhibitor kinetic marily from a comparative point of view and will studies and was confirmed by gel filtration results. probably not be generally useful as applied enzyme This would mean that there are several active sites sources. per molecule. From a sedimentation coefficient of Immobilization of choliniesterases 9.7 S, a molecular weight of 368 000 was calculated for the most highly purified BuChE of porcine Immobilized cholinesterases offer potential advan- parotid gland. The multiplicity of serum cholinester- tages for the maintenance of enzyme stability and ases, demonstrated with various mammalian species, in the detection of cholinesterase inhibitors, in stu- is probably attributable to reversible polymerization. dies of active sites, and in applications where it is The molecular activities (" turnover numbers") desired to obtain a product without using up the of some purified enzymes are given in Table 3. enzyme or having to perform additional operations to recover and reuse it (Usdin, 1970). Electrophoretic properties Immobilization can be accomplished by entrap- The serum cholinesterases have been more thor- ment of the enzyme in starch or agar gels or in oughly studied electrophoretically than have acetyl- cross-linked polyacrylamide, or by covalent fixation cholinesterases. They usually have the isoelectric PURIFICATION AND PROPERTIES OF CHOLINESTERASES 87 point at pH 3-5. However, this is dependent on the not only similar for these types of enzyme, but is electrophoretic technique used and on the source also found in the active sites from several other and purity of the preparation. Detailed electro- hydrolytic enzymes. Only recently has complete phoretic analyses of the various esterases present in amino acid analysis become available for two essen- vertebrate plasmas have been described (Augustins- tially pure cholinesterases. The results of analyses son, 1959). The isoelectric point of electric eel AChE of AChE from the electric organ of the electric eel is about pH 5. (Leuzinger & Baker, 1967) and of BuChE from the Serum BuChE is a sialo-protein whose electro- parotid gland of the pig (Tucci & Seifter, 1969) are phoretic mobility changes after treatment with siali- compared in Table 4. AChE has more valine, methio- dase (Svensmark, 1965). Horse serum BuChE, upon nine, and than has the parotid BuChE, which treatment with sialidase, gave a number of products is much richer in serine, , glycine, and with isoelectric points ranging from pH 3.6 to alanine. pH 5.2. During sialidase treatment the enzyme preserves both activity and kinetic properties. AChE Table 4 is not a sialo-protein and its electrophoretic mobility Amino acid analysis of AChE (electric organ) is not changed by sialidase treatment. and BuChE (parotid gland) Serum cholinesterases from man and a number of Residues a other mammalian species have been separated into Amino acid 2-7 distinct bands by means of different techniques AChE BuChE of gel electrophoresis. Each of these bands may be referred to as an isoenzyme. Results obtained with Asp 280 340 other techniques-e.g., gel filtration, chromatogra- Thr 112 156 phy on DEAE-cellulose, and kinetic studies of inhi- bition-also indicate isoenzymes for both human Ser 168 263 and horse serum BuChE. Glu 224 337 Holmes et al. (1968) have demonstrated, with Pro 196 225 1-naphthyl acetate as the main substrate, three Gly 196 310 groups of cholinesterase isoenzymes: a group hav- ing high molecular weight and low electrophoretic Ala 140 298 mobility; a group having intermediate molecular Val 168 118 weight and mobility; and a group having low Met 70 30 molecular weight and high mobility. As mentioned lie 84 68 above, these forms of cholinesterase may be inter- Leu 224 224 convertible. In this connexion it should be noted that variants Tyr 98 62 of the normal form of human serum BuChE have Phe 140 150 been extensively studied from a pharmacogenetic Lys 112 156 point of view (Goedde et al., 1967) and found to His 56 56 be genetically controlled. Such variants are presum- Arg 140 130 ably also present in other animal species. cysteic acid 28 27 Although there is much less evidence for the exis- tence of isoenzymes among acetylcholinesterases molecular weight 260 000 368 000 than among the serum cholinesterases, various elec- trophoretic studies indicate that they do exist. Thus, a number of isoenzymes of AChE have been identi- a Assuming 56 . fied in erythrocytes and in the brain of man, fish, and insects. Active sites Amino acid composition The nature of the active sites of cholinesterases has The amino acid composition and sequences of been determined by indirect methods and with so- the active sites of both AChE and BuChE are now called " quasi-substrates " such as 32P-labelled diiso- fairly well established. The sequence Glu-Ser-Ala is propyl phosphorofluoridate (DF32P) followed by de- 88 K.-B. AUGUSTINSSON gradation and isolation of peptides containing these the first substrate molecule reacts with AChE at a derivatives. A presentation of these results is beyond basic group on the esteratic site, and that the second the scope of this survey, but a brief statement of our substrate molecule reacts at the acidic group of the present knowledge might be appropriate. same site, forming a relatively unreactive complex. Both AChE and BuChE contain, in addition to Substrate inhibition has also been attributed to the the ester binding group (" esteratic site "), a second reaction of two molecules of substrate at the anionic active site that is negatively charged (" anionic site, which decreases sterically the ease of interaction site ") in AChE and that is a van der Waals centre with the esteratic site, leading to a decrease in in BuChE. This non-esteratic site explains why hydrolytic rates. cholinesterases are much more sensitive to quater- Brestkin et al. (1965, 1970) disagree with these nary ammonium salts than are other esterases. This interpretations. They believe that an inactive com- affinity for cationic substrates (and inhibitors) is the plex (between the acetylated enzyme and two sub- most characteristic feature of these enzymes. The strate molecules) is formed as a result of a confor- second site, which controls binding and orientation mational change in the structure of AChE. Such a of the substrate, is frequently responsible for the change does not occur when BuChE reacts with action specificity of the enzyme. Consequently, the substrates at high concentration. dominant reactive forces of this site in BuChE are There is little doubt that serine and are van der Waals forces, whereas the predominant ones the basic groups in the esteratic site of all cholin- in the anionic site of AChE are coulombic. esterases that have been studied. The strongest The existence of one (or two) additional " non- evidence for the presence of serine has been provided esteratic " site(s) has been used to explain the pheno- by degradation studies of DF32P-inhibited enzymes. menon of inhibition by excess substrate that is char- The evidence for the presence of histidine is indirect, acteristic of the acetylcholinesterases (Augustinsson, as are the indications that the acidic group of the 1948; Krupka, 1968). Since this phenomenon is esteratic site is the tyrosine hydroxyl. The carrier for not shown by BuChE, it cannot be explained merely the negative charge in the anionic site of AChE is by the existence of a second site, and must depend most likely glutamic acid. In the non-esteratic site on the type of forces between the " non-esteratic " of BuChE this aminodicarboxylic acid might be site and the substrate. It has been suggested that replaced by an acid with no free carboxyl group.

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DISCUSSION

REINER: How do you define an isoenzyme? GAGE: We have observed that the inhibition of female rat plasma by certain organophosphorus compounds AUGUSTINSSON: Isoenzymes are generally defined as closely resembles that obtained with plasma of the dog, enzymes occurring in the same organ or tissue and having the horse, and man, whereas only 20% of the cholin- the same substrate specificity and kinetic behaviour, but esterase of male rat plasma appears to be inhibited in differing in electrophoretic mobility. The term is to be this manner, the remainder being much more resistant differentiated from that of subunit. to inhibition.