Cholinesterases active site

However, the next stage turns out to be extremely slow. Despite the fact that histidine can still act as a basic catalyst, water finds it difficult to attack the carbamoyl intermediate. This step becomes the rate determining step for the whole process and the overall rate of hydrolysis of physostigmine compared to acetylcholine is forty million times slower. As a result, the cholinesterase active site becomes bunged up and is unable to react with acetylcholine. 

FIGURE 14.11 The pH activity profiles of four different enzymes. Trypsin, an intestinal protease, has a slightly alkaline pH optimnm, whereas pepsin, a gastric protease, acts in the acidic confines of the stomach and has a pH optimmn near 2. Papain, a protease found in papaya, is relatively insensitive to pHs between 4 and 8. Cholinesterase activity is pH-sensitive below pH 7 but not between pH 7 and 10. The cholinesterase pH activity profile suggests that an ionizable group with a pK near 6 is essential to its activity. Might it be a histidine residue within the active site ... 

Fig. 11.2. Schematic representation of the primary structure of secreted AChE B of N. brasiliensis in comparison with that of Torpedo californica, for which the three-dimensional structure has been resolved. The residues in the catalytic triad (Ser-His-Glu) are depicted with an asterisk, and the position of cysteine residues and the predicted intramolecular disulphide bonding pattern common to cholinesterases is indicated. An insertion of 17 amino acids relative to the Torpedo sequence, which would predict a novel loop at the molecular surface, is marked with a black box. The 14 aromatic residues lining the active-site gorge of the Torpedo enzyme are illustrated. Identical residues in the nematode enzyme are indicated in plain text, conservative substitutions are boxed, and non-conservative substitutions are circled. The amino acid sequence of AChE C is 90% identical to AChE B, and differs only in the features illustrated in that Thr-70 is substituted by Ser.

Darvesh S, McDonald RS, Darvesh KV, et al. On the active site for hydrolysis of aryl amides and choline esters by human cholinesterases. Bioorg Med Chem 2006 14(13) 4586 1599. 

We may now consider in a little more detail the interaction of true (or a-) cholinesterase with acetylcholine. Wilson and Berg mann1 suggest that there are two active sites in the enzyme, known as anionic site and esteratic site respectively. These sites (represented diagrammatically in fig. II)2 are not to be considered independent. The mode of attachment will be seen to depend upon (a) the quaternary nitrogen atom (N+< ) and... 

Because cholinesterase inhibition is a very sensitive biomarker for other chemicals, it is not always conclusive evidence of disulfoton exposure. However, depression of cholinesterase activity can alert a physician to the possibility of more serious neurological effects. Erythrocyte acetylcholinesterase activity more accurately reflects the degree of synaptic cholinesterase inhibition in nervous tissue, while serum cholinesterase activity may be associated with other sites (Goldfrank et al. 1990). In addition, a recent study showed that after rats received oral doses of disulfoton for 14 days, acetylcholinesterase levels in circulating lymphocytes correlated better with brain acetylcholinesterase activity than did erythrocyte cell cholinesterase activities during exposure (Fitzgerald and Costa 1993). However, recovery of the activity in lymphocytes was faster than the recovery of activity in the brain, which correlated better with the activity in erythrocytes. Animal studies have also demonstrated that brain acetylcholinesterase depression is a sensitive indicator of neurological effects (Carpy et al. 1975 Costa et al. 1984 Schwab and Murphy 1981 Schwab et al. 1981, 1983) however, the measurement of brain acetylcholinesterase in humans is too invasive to be practical. 

Methyl parathion itself is not a strong cholinesterase inhibitor, but one of its metabolites, methyl paraoxon, is an active inhibitor. Methyl paraoxon inactivates cholinesterase by phosphorylation of the active site of the enzyme to form the dimethylphosphoryl enzyme. Over the following 24-48 hours there is a process, called aging, of conversion to the monomethylphosphoryl enzyme. ... 

Reversible cholinesterase inhibitors form a transition state complex with the enzyme, just as acetylcholine does. These compounds are in competition with acetylcholine in binding with the active sites of the enzyme. The chemical stracture of classic, reversible inhibitors physostigmine and neostigmine shows their similarity to acetylcholine. Edrophonium is also a reversible inhibitor. These compounds have a high affinity with the enzyme, and their inhibitory action is reversible. These inhibitors differ from acetylcholine in that they are not easily broken down by enzymes. Enzymes are reactivated much slower than it takes for subsequent hydrolysis of acetylcholine to happen. Therefore, the pharmacological effect caused by these compounds is reversible. 

Because atropine has never been found to have reactivating activity in vitro, the reactivation that occurred in the rats treated with atropine sulfate is assumed to be spontaneous. It is apparent from Table 3 that addition of 2-PAM I to atropine increased cholinesterase reactivation by 40.5% in the parotid gland, by 127.8% in the gastrocnemius muscle, and by 8.2% in the brain. The especially large change in cholinesterase activity in skeletal muscle suggests that this may be the principal site at which 2-PAM I antagonizes inhibition of cholinesterase. 

General supportive care should be provided as outlined above. Extra precautions should be taken to ensure that rescuers and health care providers are not poisoned by exposure to contaminated clothing or skin. This is especially critical for the most potent substances such as parathion or nerve gas agents. Antidotal treatment consists of atropine and pralidoxime (see Table 58-4). Atropine is an effective competitive inhibitor at muscarinic sites but has no effect at nicotinic sites. Pralidoxime given early enough is capable of restoring the cholinesterase activity and is active at both muscarinic and nicotinic sites. 

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. 

Inhibition of the cholinesterase enzymes depends on blockade of the active site of the enzyme, specifically the site that binds the ester portion of acetylcholine (Fig. 7.48). The organophosphorus compound is thus a pseudosubstrate. However, in the case of some compounds such as the phosphorothionates (parathion and malathion, for example), metabolism is necessary to produce the inhibitor. 

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. 

Earlier work in this field [28] indicated that acetylcholinesterase enzymes would be suitable biomolecules for the purpose of pesticide detection, however, it was found that the sensitivity of the method varied with the type and source of cholinesterase used. Therefore the initial thrust of this work was the development of a range of enzymes via selective mutations of the Drosophila melanogaster acetylcholinesterase Dm. AChE. For example mutations of the (Dm. AChE) were made by site-directed mutagenesis expressed within baculovirus [29]. The acetylcholinesterases were then purified by affinity chromatography [30]. Different strategies were used to obtain these mutants, namely (i) substitution of amino acids at positions found mutated in AChE from insects resistant to insecticide, (ii) mutations of amino acids at positions suggested by 3-D structural analysis of the active site,... 

Acetylcholinesterase is only cholinesterase in insects. It is mainly located in the neuropile (area of synapses between nerve fibers) of the CNS in insects (Toutant, 1989). AChE contains two active sites, the esteratic site and the anionic site. The esteratic site possesses the hydroxyl group of serine and a basic nucleophilic imidazole group of histidine. The anionic site has a free carboxyl group (aspartic acid and/or glutamic acid). The interaction of ACh with AChE can be divided into three steps, as shown in Figure 7.13. The first... 

Saxena, A., Redman, A.M.G., Jiang, X., Lockridge, O., Doctor, B.P. (1999). Differences in active-site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase. Chem. Biol. Interact. 119-20 61-9. 

The covalent bond between OP and the active site serine of intact cholinesterase is stable, but not irreversible. Hydrolysis can occur with a half-life of between 10 and 35,000 min, depending on the enzyme, OP, temperature, pH, and buffer composition. The adduct becomes irreversibly bound to the enzyme after one of the alkyl groups on the OP is lost in a step called aging (Benschop and Keijer, 1966 Michel et al., 1967). The dealkylated OP makes a stable salt bridge with the protonated histidine of the catalytic triad, so that histidine is no longer available for the dephosphorylation step that would otherwise have restored the enzyme to an uninhibited state. Hundreds of scientists have contributed to this understanding... 

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