Cholinesterases substrate inhibition

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

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. 

Benzoylcholine is a substrate of pseudo-cholinesterase but not of the true cholinesterase. It inhibits the true cholinesterase of man (laked red cells centrifuged and the supernatant liquid diluted 1/150, acetylcholine substrate 0 005 m) from 30 per cent at a concentration of 0-1 M to 85 per cent at a concentration of 0-3 M. Benzoylcholine injected intravenously into rabbits will, at a dose of 8-14 mg./kg. body weight, produce a head drop lasting 40-120 sec. 

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. 

Scott (S12) purified usual serum cholinesterase and cholinesterase from a homozygous silent individual having about 2 % of usual activity, which corresponds to silent type II cholinesterase. In most respects, the properties of the two purified cholinesterases were similar but not identical. The greatest differences noted were that the silent enzyme was more heat stable, and that, with it, there was less substrate inhibition by excess ben-zoylcholine. 

At high substrate concentrations, neither human nor horse plasma cholinesterase shows substrate inhibition with either acetyl- or butyryl-choline, but substrate inhibition is observed with halogenoacetylcholines... 

Main (M4), in an excellent review article on cholinesterase inhibitors, discussed the additional complication of substrate inhibition in the above general mechanism. However, in many inhibition studies Michaelis-Menton kinetics are obeyed quite closely. One simple reaction scheme which leads to such kinetics is as follows ... 

Note that the kinetic interactions of substrates with AChE and BuChE are in reality more complex than portrayed in Fig. 5. Both cholinesterases have been shown to display substrate inhibition and activation, depending on the incubation condilion.s (Masson et ai., 2004). probably as a result of the presence of a binding site separate from the active site, termed the peripheral anionic site (Changeux. 1966 Taylor and Radic, 1994 Barak et ai., 1995 Soreq and Seidman,... 

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. 

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 ... 

A number of substituted p-aminoacetates inhibit the enzyme cholinesterase. The main function of this enzyme is to hydrolyze acetyl choline and thereby terminate the action of that substrate as a neurotransmitter. Such inhibition is functionally equivalent to the administration of exogenous acetylcholine. Direct administration of the neurotransmitter substance itself is not a useful therapeutic procedure due to rapid drug destruction and unacceptable side... 

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... 

Fig. 12. Progress curve of inhibition of horse-serum cholinesterase by eserine and by di -isopropyl phosphorofluoridate in the absence of a substrate at pH 7-4 and 20°. x---x, 5x 10 8 M eserine O—O.ca. 3 x 10-10 M di-isopropyl phosphorofluoridate.

Fig. 13. Effect of substrate concentration on inhibition of horse-serum cholinesterase.1 Enzyme activity was estimated by titration with 0-01 n NaOH at pH 7-4 and 20°. — , control, no inhibitor x — x, 2x 10 7 m eserine 0—O, 5 x 10 8 m di-isopropyl phosphorofluoridate.

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. 

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. 

The pesticides are detected electrochemically by measuring the degree of inhibition of cholinesterase on the screen-printed electrodes. The degree of inhibition can be thought of as the ratio of the response of electrodes (to substrate) exposed to pesticide (standard or wool extract) to that of electrodes not exposed to pesticide or exposed to a blank (extract from wool which has not been exposed to pesticide). An efficient and convenient way of doing this is to expose the SPCEs to standards and wool extracts prior to measurement of inhibition in the electrochemical cell. 

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