Acetylcholinesterase acidic group

Although OPPs and carbamates exhibit very similar modes of action in various animal species, i.e, acetylcholinesterase inhibition in the CNS with resulting paralysis—there is an important difference between the two classes of pesticides. Carbamates do not require metabolic conversion prior to exhibiting their toxicity. Furthermore the enzyme activity may at times be rapidly regenerated by reversal of inhibition. The kinetics of the inhibition (carbamoylation) reaction have been well studied in it electrophilic carbamoyl moieties form covalent bonds with enzyme esteratic sites. This is followed by carbamate transfer of an acidic group to the site to yield the acetylated enzyme complex (ref. 176). 

Most irreversible enzyme inhibitors combine covalently with functional groups at the active sites of enzymes. These inhibitors are usually chemically reactive, and many of them show some specificity in terms of the amino acid groups which they react with. Diisopropyl fluorophosphate (DFP), for example, forms a covalent adduct with active site serine residues, such as in the serine proteases, and in acetylcholinesterase, which explains its toxic effect on animals. Irreversible enzyme inhibition can be used to identify important active site residues. A special case of irreversible enzyme inhibition is the effect of suicide inhibitors, which are generally chemically unreactive compounds that resemble the substrate of the target enzyme and bind at the active site. The process of enzyme turnover begins, but the inhibitor is so... 

Figure 9.2 Chemical reaction of OPj with the enzyme acetylcholinesterase (Ser, serine His, histidine Glu, glutamate Trp, tryptophan X, acid group)...

The phosphorus ester molecule, blocking the activity of acetylcholinesterase, first takes up a steric orientation determined by the anionic and acidic groups of the enzyme, and this is then followed by hydrolysis of the phosphorus ester and by phosphorylation of the hydroxyl group of serine at the esteratic site of the enzyme ... 

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

So far, many kinds of nucleophiles active for hydrolysis such as imidazolyl-, amino-, pyridino-, carboxyl- and thiol-groups, have been used for preparation of hydrolase models. Overberger et al.108,1091 prepared copolymers of vinylimidazole and acrylic acid 60 (PVIm AA), by which the cationic substrate, 61 (ANTI), was hydrolyzed. This kind of copolymer is considered to be a model of acetylcholinesterase. With ANTI, the rate of the copolymer catalysis was higher than that of imidazole itself in the higher values of pH, as is seen in Table 9. In this work, important contributions of the electrostatic interactions are clear. The activity of the copolymer was not as high with the negatively charged and neutral substrates. 

However, not included in the above mechanisms are other amino acid side-chains at the active site, whose special role will be to help bind the reagents in the required conformation for the reaction to occur. Examples of such interactions are found with acetylcholinesterase and chymotrypsin, representatives of a group of hydrolytic enzymes termed serine hydrolases, in that a specific serine amino acid residue is crucial for the mechanism of action. 

Selective bioactivation (toxification) is illustrated in the case of the insecticide malathion (3.35). This acetylcholinesterase inhibitor is desulfurized selectively to the toxic malaoxon, but only by insect and not mammalian enzymes. Malathion is therefore relatively nontoxic to mammals (LDjg = 1500 mg/kg, rat p.o.). Higher organisms rapidly detoxify malathion by hydrolyzing one of its ester groups to the inactive acid, a process not readily available to insects. This makes the compound doubly toxic to insects since they cannot eliminate the active metabolite. 

Irreversible inhibitors combine or destroy a functional group on the enzyme so that it is no longer active. They often act by covalently modifying the enzyme. Thus a new enzyme needs to be synthesized. Examples of irreversible inhibitors include acetylsal-icyclic acid, which irreversibly inhibits cyclooxygenase in prostaglandin synthesis. Organophosphates (e.g., malathion, 8.10) irreversibly inhibit acetylcholinesterase. Suicide inhibitors (mechanism-based inactivators) are a special class of irreversible inhibitors. They are relatively unreactive until they bind to the active site of the enzyme, and then they inactivate the enzyme. 

In the acylation step a nucleophilic group on one of the amino-acid side chains at the active site behaves as the nucleophile. As we have seen in Section 25-9B, the nucleophile of carboxypeptidase is the free carboxyl group of glutamic acid 270. In several other enzymes (chymotrypsin, subtilisin, trypsin, elastase, thrombin, acetylcholinesterase), it is the hydroxyl group of a serine residue ... 

After the challenge of the dynamic thiolester system (CDS-1 A) with acetylcholinesterase, the optimal constituents were immediately identified and hydrolyzed to acid and thiol products. The thiol product formed was simultaneously incorporated in the dynamic system to regenerate the optimal thiolester. During the acetylcholinesterase resolution process, two acid products, acetic and propionic acid, respectively, were mainly detected, with the acetyl ester hydrolyzed more rapidly (tl/2 = 210 min) than the propionyl ester (t1/2 = 270 min) as shown in Fig. 2. Only after significant hydrolysis of these two acyl species did the enzyme start to hydrolyze slowly the butyrate thiolester (t1/2 = 1,100 min), a lag phase possibly caused by inhibitory activities of the present thiolesters [8]. All the other acyl groups remained untouched by enzyme, a result which is in accordance with the known specificity of acetylcholinesterase. 

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... 

Applications. A biotinylated GOX-based biosensor was developed based on a new electropolymerized material consisting of a pol3rp3uidyl complex of ruthenium(II) functionalized with a pyrrole group [90]. Because histidine, lysine and arginine functions also coordinate Os /Os , biosensors based on co-electrodeposited GOX, HRP, soybean peroxidase (SBP) and laccase with redox Os /Os polymer have been developed [89]. A metal chelate formed by nickel and nitrilotriacetic acid was used to modify a screen-printed electrode surface. The functionalized support allowed stable attachment of acetylcholinesterase and the resulting biosensor was used for sensitive detection of organophosphorus insecticides [91]. This method is attractive because it ensures a controlled and oriented enzyme immobilization, considerably improving the sensitivity and the detection limit. 

Clavulanic acid is a mechanism-based irreversible inhibitor and could be classed as a suicide substrate (Chapter 4). The drug fits the active site of (3-lactamase and the 13-lactam ring is opened by a serine residue in the same manner as penicillin. However, the acyl-enzyme intermediate then reacts further with another enzymic nucleophilic group (possibly NH2) to bind the drug irreversibly to the enzyme (Fig. 10.54). The mechanism requires the loss or gain of protons at various stages and an amino acid such as histidine present in the active site would be capable of acting as a proton donor/acceptor (compare the mechanism of acetylcholinesterase in Chapter 11). 

There are three primary cholinesterases (ChE) in the body. Acetylcholinesterase is located in the vicinity of ACh receptors at neuronal and neuromuscular junctions. Acetylcholinesterase terminates ACh activity via hydrolysis into choline and acetic acid. The positively charged choline portion of the ACh molecule attaches to the anionic site and the acetyl region attaches to the esteratic site on the AChE molecule. Eollowing the attachment of the two regions, choline is rapidly released to be recycled back into the presynaptic nerve terminal, and the acetyl group reacts with... 

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