Cholinesterases substrate specificity

Cholinesterases (ChEs), polymorphic carboxyles-terases of broad substrate specificity, terminate neurotransmission at cholinergic synapses and neuromuscular junctions (NMJs). Being sensitive to inhibition by organophosphate (OP) poisons, ChEs belong to the serine hydrolases (B type). ChEs share 65% amino acid sequence homology and have similar molecular forms and active centre structures [1]. Substrate and inhibitor specificities classify ChEs into two subtypes ... 

Cholinesterases secreted by parasitic nematodes of (predominantly) the alimentary tract or other mucosal tissues are authentic AChEs when analysed by substrate specificity, inhibitor sensitivities and primary structure. In the first two respects, they resemble vertebrate AChEs, whereas somatic (and therefore presumably neuronal) enzymes of nematodes analysed to... 

Cholinesterases are widely distributed throughout the body in both neuronal and non-neuronal tissues. Based largely on substrate specificity, the cholinesterases are subdivided into the acetylcholinesterases (AChEs) (EC... 

Cholinesterases are subdivided into acetylcholinesterase and cholinesterase, one with a narrow, the other with broad substrate specificity [109-112], Both enzymes exist in multiple molecular forms distinguishable by their subunits association (Fig. 2.4). The hydrodynamic properties of these associations have allowed globular (G) and asymmetric (A) forms to be distinguished. The G forms can be hydrophilic (water-soluble, and excreted into body fluids) or amphiphilic (membrane-bound). The homomeric class exists... 

The foregoing studies have dealt chiefly with model substrates in vitro. Several of the early papers by Augustinsson, referred to in Section 4.1.1, considered substrate specificity from the viewpoint of species variations. It is also important to recognize that plasma cholinesterase may be associated with the hydrolysis, in vivo, of a large number of drugs (K4, LI, L4) that contain ester bonds susceptible to enzymic hydrolysis. Apart from succinylcholine (Section 3.1), cholinesterase is known to be responsible in man for the hydrolysis of cocaine (S40), procaine (K2), and other esters with local anesthetic properties. Whether enzymatic hydrolysis terminates the pharmacologic effect depends on the whole mechanism of action of the particular drug. 

In spite of an immense literature on the substrate specificity of cholinesterase, the significance of its variation from one species to another (e.g., A21, A27, M25, E2) remains obscure. Even such closely related species as the Macaque and Mangabey monkeys show striking differences in the substrate specificities of their plasma cholinesterases (A27). There is, however, little variation in substrate specificity for the enzyme when it occurs in different tissues within a species. The complications in this field were indicated early on by Kalow and his associates, who showed that the substrate affinity of the usual human plasma cholinesterase is quite different from that of the atypical enzyme (DIO). It is therefore highly desirable to state the source of the enzyme, as well as the substrate used, when describing work on plasma cholinesterase. 

Horse and human plasma cholinesterases have been found to have similar substrate specificity profiles (M25), for example, in respect to the relative rates of hydrolysis of some isomeric substrates (B14), as summarized in Table 4. It can be assumed that the areas in the regions of the... 

Main et al. (M5) found high concentrations of a butyrylcholine esterase, of relative molecular mass 83,000 daltons, in pooled rabbit serum. This was unexpected, since rabbit serum is classed with those mammalian sera that have low cholinesterase activities (A25, E2). Substrate specificity confirmed that the enzyme was a butyrylcholine esterase. Moreover, the active site concentration of the enzyme was five times that found for pooled horse serum, which is a rich source of the enzyme. The known fact that some rabbits can metabolize atropine, whereas others are unable to do so, can be explained by the presence or absence of serum atropinase, which is a genetic trait. Perhaps the subunit-sized butyrylcholine esterase is characteristic of some rabbits, but absent in others This seems probable according to Ellis (E9) and Koelle... 

The effectiveness of a given inhibitor of the enzyme, as with substrate specificity, varies with the source of cholinesterase. values, defined as the molar concentration of inhibitor giving 50% enzymic activity, are often used to compare the potency of inhibitors. By no means, however, do all investigators specify the experimental conditions under which their measurements were made. It is therefore advisable to use 50 values only as crude indices of inhibitor potency. Differences between the reactivities of horse serum isoenzymes with organophosphates have been reported (C6, R3). Inhibition of horse serum cholinesterase is stereospecific (B28), and thus the enzyme reacts with optically pure (—)-sarin at least 4000 times faster than with ( + )-sarin. 

Differences between the species toward the thiocholine substrates have been reported. Table 11.1 illustrates some observed differences with three species. For plasma pseudocholinesterase measurements, dog, rabbit, and man show higher substrate specificity for butyryl substrates, whereas rat, mouse, and hamster show higher specihcity for propionyl substrates all the species show less specihcity toward benzoyl substrates. Female rats have higher values compared to males with all three substrates, and the cholinesterase levels in platelets are higher in rats compared to the very low levels in human platelets. 

The two cholinesterase enzymes, acetyl (AChE) and butyryl (BuChE), although closely related, show differences both in their occurrence in the body (leading to their older vernacular names of erythrocyte, red cell, or true cholinesterase in the case of AChE and plasma or pseudo-cholinestcrase for BuChE) and in their substrate specificity. AChE is more correctly called acetylcholine acetylhy-drolase (EC 3.1.1.7), and BuChE is more correctly called acylcholinc acylhydrola.se (EC 3.1.18). AChE is present in most vertebrates in several molecular forms, whereas BuChE is present in only c te, the tctramcric T form (Massoulie, 2002). 

True and pseudo-cholinesterase. The above serum preparations contained both the true and pseudo- cholinesterases of Mendel and Rudney.1 The effect of di-isopropyl phosphorofluoridate on these components was examined separately by means of the specific substrates described by Mendel, Mundel and Rudney,2 using the titration method described above. Phosphorofluoridate (5 x 10 8m) gave an inhibition of 57 per cent of the activity towards 00045m acetylcholine, 30 per cent of the activity towards 0-0005 m acetyl-/ methyl-choline, and 40 per cent of that towards 0-005 m benzoylcholine, after incubating the enzyme with the poison for 5 min. Thus in these experiments there appeared to be no appreciable difference in sensitivity of the true and pseudo-cholinesterases of horse serum to phosphorofluoridates. 

The cholinesterases, acetylcholinesterase and butyrylcholinesterase, are serine hydrolase enzymes. The biological role of acetylcholinesterase (AChE, EC 3.1.1.7) is to hydrolyze the neurotransmitter acetylcholine (ACh) to acetate and choline (Scheme 6.1). This plays a role in impulse termination of transmissions at cholinergic synapses within the nervous system (Fig. 6.7) [12,13]. Butyrylcholinesterase (BChE, EC 3.1.1.8), on the other hand, has yet not been ascribed a function. It tolerates a large variety of esters and is more active with butyryl and propio-nyl choline than with acetyl choline [14]. Structure-activity relationship studies have shown that different steric restrictions in the acyl pockets of AChE and BChE cause the difference in their specificity with respect to the acyl moiety of the substrate [15]. AChE hydrolyzes ACh at a very high rate. The maximal rate for hydrolysis of ACh and its thio analog acetyl-thiocholine are around 10 M s , approaching the diffusion-controlled limit [16]. 

Certain therapeutic effects can be attributed to the inhibition of specific enzymic reactions. The inhibition of cholinesterase (Section 1.06.3), orotidylate pyrophosphorylase (Section 1.06.5) and of dihydrofolate reductase (Section 1.06.6.3) have already been discussed. They illustrate two modes of action, chemical alteration of the enzyme and competition with a substrate for the active site. 

Special interest adheres to the group of cholinesterases (ChE), not only in view of their physiological role in conductive tissues, but also because their specific behavior towards substrates and inhibitors and their high efficiency towards cationic substrates permit exact kinetic measurements. In spite of an enormous amount of experimental work, the exact structure of the active surface of cholinesterases is still controversial [see the review of Whittaker (/)]. The following representation will discuss the results already achieved and point out the many problems in this field still awaiting solution. 

The extensive studies on substrate and inhibitor specificity, on kinetics of hydrolysis, and on the influence of pH variations on the reactions catalyzed by cholinesterases have given very instructive information on the structure of the active surface and the mechanism of enzymatic hydrolysis. The conclusions reached in the various chapters of the present dis-... 

In contrast to acetylcholinesterase, which is selective for acetylcholine, butyryl-cholinesterase tolerates a wider variety of esters and is more active with butyryl-and propionylcholines than acetylcholine [7]. Structure-activity relationship studies have shown that different steric restrictions in the acyl pockets of AChE and BChE cause the difference in specificity to the acyl moiety of the substrate [6]. 

Acetylcholinesterase (AChE) (also termed true cholinesterase ) is found in the synaptic cleft of cholinergic synapses, and is of undoubted importance in regulation of neurotransmission by rapid hydrolysis of released endogenous acetylcholine (ACh). AChE is also found in erythrocytes and in the CSF, and can be present in soluble form in cholinergic nerve terminals, but its function at these sites is not clear, AChE is specific for substrates that include acetylcholine and the agents methacholine and acetylthiocholine. but it has little activity with other esters. It has a maximum turnover rate at very low concentrations of AChE (and is inhibited by high concentrations). 

Enzyme inhibitors are species that cause a decrease in the activity of an enzyme. Inhibitors usually interact with the enzyme itself, forming enzyme-inhibitor (E I) complexes, but in a few cases, the inhibition mechanism involves reaction with one of the substrates. Inhibition is considered to be reversible if the enzyme recovers its activity when the inhibitor is removed, and irreversible if the inhibitor causes a permanent loss of activity. Reversible inhibition affects the specific activity and apparent Michaelis-Menten parameters for the enzyme, while irreversible inhibition (where the E I complex formation is irreversible) simply decreases the concentration of active enzyme present in the sample. A well-known example of irreversible inhibition is the effect of nerve gas on the enzyme cholinesterase. 

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