Cholinesterases substrate activation

Reiner, F., Simeon-Rudolf, V. (2000). Cholinesterase substrate inhibition and substrate activation. Pflugers Arch. Eur. J. Physiol. 440 R118-20. 

Mosca A, Bonora R, Ceriotti F, Franzini C, Lando G, Patrosso MC, et al. Assay using succinyldithiochohne as substrate the method of choice for the measurement of cholinesterase catalytic activity in serum to diagnose sucdnylchoMne sensitivity. Clin Chem Lab Med 2003 41 317-22. 

Fig. 13. Effect of substrate concentration on inhibition of horse>sermn cholinesterase. Enzyme activity was estimated by titration with 0-01 n NaOH at pH 7-4 and 20°. — , control, no inhibitor x — x, 2x 10 m eserine 0—O, 5 X 10 M di-isopropyl phosphorofiuoridate.

A number of papers describe tedmiques for determination of cholinesterase activity based on amperometric measurement of products formed as a result of enzymatic hydrolysis (equation 1). In this case, artificial (butyryl or acetyl thiocholine) cholinesterase substrates are used. Thiocholine, formed as a result of cholinesterase-catalyzed hydrolysis can be measured amperometrically on a platinum electrode (14, 15) or mercury electrode (16). Analyses based on thiocholine determination employing an electrode modified by cobalt phthalocyanine (17-22) or cobalt tetraphenylporphyrin (23) have been described. Enzymatic hydrolysis of... 

Organophosphates, thiocarbamates, carbamates, carbamoyloximes, dithiocarbamates, and ureas were included among 100 pesticides and metabolites detected on TLC plates by their cholinesterase inhibiting properties. After developing the plates in ether, xylene, di-n-butyl ether, n-butyl acetate or methyl-isobutyl ketone, the plates were exposed to bromine vapor. The compounds were oxidized to their oxo derivatives, which exhibited more effective cholinesterase inhibitory activity. Bovine liver suspension served as enzyme source and the substrates 2-naphthyl acetate and Fast Blue B salt as the chromogenic agents (163a). 

Cholinesterases are the prime example of enzymes that have been found to be subject to substrate modulation. Specifically, acetylcholinesterase is known to experience substrate inhibition and butyiylcholinesterase is subject to substrate activation. To model these effects, equation 23 (Reiner Simeon-Rudolf 2000) has been used. 

Enzyme-catalysed reactions are widely used for analytical purposes, for the determination of substrates (e.g. glucose oxidase for determination of glucose) and of inhibitors (such as pesticides, by their inhibition of cholinesterase) and activators. Although enzymes are very useful as analytical reagents, they are not classified individually in this Dictionary. However, enzymes themselves are extensively assayed by clinical chemists, biochemists, forensic scientists and food chemists, and the substrates used for such assays are carefully chosen to achieve optimum sensitivity, selectivity and reliability. Such substrates are listed in this Dictionary, as are the co-enzymes (co-factors) required by many redox enzymes, for example nicotinamide adenine dinucleotide (NAD /NADH) which is a co-enzyme for many dehydrogenases, e.g. 

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 ... 

Procedure Cholinesterase activity in analyzed tissue or the matrix (biotest with immobilized AChE) is determined in the incubation media [consisting of substrate ATCh - 34 mmol maleate buffer 0.1 M, pH = 6.0- 6.5 ml sodium citrate 0.1 M - 0.5 ml CuS045H20 0.03M -1.0 ml distilled H20 (or inhibitor in variant with toxin analyzed) -1.0 ml potassium ferricyanide 0.005 M -1 ml.] Volume of incubation media in one test - 400 mcl. As a blank (control sample), a treatment of the exposure without the substrate is used. If inhibitory effects of allelochemical (or any toxin) are analyzed, before the substrate addition the sample was preliminary exposed to allelochemical inhibitor. Two methods for the AChE-biotests may be recommended (i) in microcells ( stationary conditions ) and (ii) in flowing columns-reactors ( dynamic conditions ). 

Observations The preliminary treatment of the cholinesterase-containing material with allelochemical (or other compound, e.g. active oxygen species, ozone free radicals and peroxides, formed in allelopathic relations) is for 30 min, then a substrate acetylcholinesterase is added to the reaction medium and final reaction of hydrolysis is for 1 h. 

Organophosphate and carbamate pesticides are potent inhibitors of the enzyme cholinesterase. The inhibition of cholinesterase activity by the pesticide leads to the formation of stable covalent intermediates such as phosphoryl-enzyme complexes, which makes the hydrolysis of the substrate very slow. Both organophosphorus and carbamate pesticides can react with AChE in the same manner because the acetylation of the serine residue at the catalytic center is analogous to phosphorylation and carbamylation. Carbamated enzyme can restore its catalytic activity more rapidly than phosphorylated enzyme [17,42], Kok and Hasirci [43] reported that the total anti-cholinesterase activity of binary pesticide mixtures was lower than the sum of the individual inhibition values. 

In AChE-based biosensors acetylthiocholine is commonly used as a substrate. The thiocholine produced during the catalytic reaction can be monitored using spectromet-ric, amperometric [44] (Fig. 2.2) or potentiometric methods. The enzyme activity is indirectly proportional to the pesticide concentration. La Rosa et al. [45] used 4-ami-nophenyl acetate as the enzyme substrate for a cholinesterase sensor for pesticide determination. This system allowed the determination of esterase activities via oxidation of the enzymatic product 4-aminophenol rather than the typical thiocholine. Sulfonylureas are reversible inhibitors of acetolactate synthase (ALS). By taking advantage of this inhibition mechanism ALS has been entrapped in photo cured polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ) to prepare an amperometric biosensor for... 

Effect of substrate concentration. In the following experiments the cholinesterase activities were measured by a continuous titration method. The digest of acetylcholine and horse-serum cholinesterase (total vol. 10 ml.), containing bromothymol blue and 0-0002 m phosphate, was titrated with 0-01 n NaOH to maintain the pH at 7-4. The titrations, which were carried out at 20°, were linear over a period of 10-15 min. The velocity was expressed as ml. 0-01 n NaOH/5 min. under the conditions used, it was proportional to the enzyme concentration. When an inhibitor was added, this was equilibrated with the enzyme, etc., for 5 min. at 20° before adding the substrate contained in a volume of 1 ml. 

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. 

Semm albumin is not an enzyme but a transport protein, yet it has demonstrated hydrolytic activity against a variety of xenobiotic substrates. This este-rase-like activity has been known for years, but there is still confusion in the literature regarding its nature and mechanism. Indeed, it was not clear whether this activity is intrinsic to the albumin molecule or results from contamination of albumin preparations by one or more hydrolytic enzymes. More-recent studies with highly purified human serum albumin (HSA) have confirmed that the protein has an intrinsic esterase activity toward several substrates, but that activity due to contaminants and particularly semm cholinesterase is involved... 

Whereas the above evidence clearly points to a catalytic activity of serum albumin, it does not exclude an activity toward less-reactive substrates due to contamination of some HSA preparations. Indeed, the hypothesis of a contamination by plasma cholinesterase (EC 3.1.1.8) has been raised [126][127]. The efficient hydrolysis of nicotinate esters by HSA (see Chapt. 8) [128][129] could be due to contamination by cholinesterase in samples of a commercially available, essentially fatty acid free albumin. Support for this hypothesis was obtained when HSA contaminated with cholinesterase was resolved into two peaks by affinity chromatography, and the esterase activity toward nicotinate esters was found exclusively in the cholinesterase fraction [130],... 

Thioesters play a paramount biochemical role in the metabolism of fatty acids and lipids. Indeed, fatty acyl-coenzyme A thioesters are pivotal in fatty acid anabolism and catabolism, in protein acylation, and in the synthesis of triacylglycerols, phospholipids and cholesterol esters [145], It is in these reactions that the peculiar reactivity of thioesters is of such significance. Many hydrolases, and mainly mitochondrial thiolester hydrolases (EC 3.1.2), are able to cleave thioesters. In addition, cholinesterases and carboxylesterases show some activity, but this is not a constant property of these enzymes since, for example, carboxylesterases from human monocytes were found to be inactive toward some endogenous thioesters [35] [146], In contrast, allococaine benzoyl thioester was found to be a good substrate of pig liver esterase, human and mouse butyrylcholinesterase, and mouse acetylcholinesterase [147],... 

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]. 

Acetylcholinesterase can be inhibited by two general mechanisms. In the first mechanism, positively charged quaternary ammonium compounds bind to the anionic site and prevent ACh from binding—a simple competitive inhibition. In the second mechanism, the agents act either as a false substrate for the cholinesterase or directly attack the esteratic site in both cases they covalently modify the esteratic site and non-competitively prevent further hydrolytic activity. Either mechanism can be effective in preventing the hydroly-... 

Various esterases exist in mammalian tissues, hydrolyzing different types of esters. They have been classified as type A, B, or C on the basis of activity toward phosphate triesters. A-esterases, which include arylesterases, are not inhibited by phosphotriesters and will metabolize them by hydrolysis. Paraoxonase is a type A esterase (an organophosphatase). B-esterases are inhibited by paraoxon and have a serine group in the active site (see chap. 7). Within this group are carboxylesterases, cholinesterases, and arylamidases. C-esterases are also not inhibited by paraoxon, and the preferred substrates are acetyl esters, hence these are acetylesterases. Carboxythioesters are also hydrolyzed by esterases. Other enzymes such as trypsin and chymotrypsin may also hydrolyze certain carboxyl esters. 

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. 

In this respect, the most important information has been obtained from the effect of pH changes on enzymic activity. In Fig. 2 the pH-activity curves are represented for true cholinesterase (from Torpedo marmorata) and pseudo-ChE (from human serum), with ACh as substrate. The two curves are not only similar to each other, but also to the curves, characteristic for other, unspecific esterases (37). For the correct interpretation of such curves, it is important to make sure that only the protein in the... 

The second pK, 8.5-9.5, derived from the pH-activity curves, is much more difficult to interpret. This pK is naturally absent in the system imidazol + ester (21). It is also subject to much greater variation than pK0. This has been demonstrated for a variety of substrates (Fig. 3), but is especially prominent when thiol esters are being studied (Figs. 4 and 5). In the system eel esterase-acetylthiocholine, no decrease of activity is observed on the alkaline side up to pH 11, and for plasma cholinesterase-acetylthiocholine the decrease is very much delayed, when compared with the oxy ester, acetylcholine (see Fig. 2). Similar observations have been made with other esterases and other thiol esters (44)- They indicate that the second component 02 of the esteratic site, to which pK has to be ascribed, may be less essential for certain substrates than for others. 

Fig. 5. pH-activity curve of the system human plasma cholinesterase-acetyl-thiocholine. Substrate concentration 3 X 10-2 M. 

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-... 

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