The active site of acetylcholinesterase

The design of anticholinesterases depends on the shape of the enzyme active site, the binding interactions involved with acetylcholine, and the mechanism of hydrolysis. 

There are two important areas to be considered—the anionic binding site and the ester binding site (Fig. 11.46). 

The histidine residue acts as an acid/base catalyst throughout the mechanism, while serine plays the part of a nucleophile. This is not a particularly good role for serine since an aliphatic alcohol is a poor nucleophile. In fact, serine by itself is unable to hydrolyse an ester. However, the fact that histidine is close by to provide acid/base catalysis overcomes that disadvantage. There are several stages to the mechanism. 

Stage 1. Acetylcholine approaches and binds to the acetylcholinesterase enzyme. The histidine residue acts as a base to remove a proton from the serine hydroxyl group, thus making it strongly nucleophilic. Nucleophilic addition to the ester takes place and opens up the carbonyl group. 

Stage 2. The carbonyl group reforms and expels the alcohol portion of the ester (i.e. choline). This process is aided by histidine which now acts as an acid catalyst by donating a proton to the departing alcohol. 

---

Fish rapidly absorb, metabolize, and excrete chlorpyrifos from the diet (Barron etal. 1991). The mechanism of action of chlorpyrifos occurs via phosphorylation of the active site of acetylcholinesterase after initial formation of chlorpyrifos oxon by oxidative desulfuration. In studies with channel catfish (Ictalurus punctatus), the oral bioavailability of chlorpyrifos was 41%, substantially higher than in mammals. Catfish muscle contained less than 5% of the oral dose with an... 

The organophosphorus (OP) and carbamate insecticides are used to control a wide variety of insect pests. The acute toxicity of the OPs and carbamates varies, and many of them have high mammalian toxicity. These compounds react chemically with the active site of acetylcholinesterase, producing a blocked enzyme that cannot degrade acetylcholine. The concentration of acetylcholine then builds up and hyperexcitation occurs. The signs of intoxication include resdessness, tremors, convulsions, and paralysis. Blockage of acetylcholinesterase by OPs is persistent, and recovery of the enzyme takes many hours or even days. The mode of action of the carba-... 

E. Elhanany, A. Ordentlich, O. Dgany, D. Kaplan, Y. Segall, R. Barak, B. Velan and A. Shafferman, Resolving pathways of interaction of covalent inhibitors with the active site of acetylcholinesterases MALDI-TOF/MS analysis of various nerve agent phosphyl adducts, Chem. Res. Toxicol., 14, 912-918 (2001). 

Mechanism of action Isoflurophate [eye soe FLURE oh fate] (diisopropylfluorophosphate, DFP) is an organophosphate that covalently binds to a serine-OH at the active site of acetylcholinesterase (Figure 4.9). Once this occurs, the enzyme is permanently inactivated, and restoration of acetylcholinesterase activity requires the synthesis of new enzyme molecules. Following covalent modification of acetylcholinesterase, the phosphorylated enzyme slowly releases one of its isopropyl groups (Figure 4.9). The loss of an alkyl group, which is called... 

In a discovery project that is reminiscent of the discovery of captopril, scientists at Takeda created a hypothetical structure for the active site of acetylcholinesterase, based on SAR from previous biochemical and medicinal chemical work (141). The model consisted of (in addition to the serine protease-like catalytic machinery) an anionic binding site separating two discrete hydrophobic binding sites. This model was then used to design inhibitors of the enzyme (reviewed i n ref. 142). One set of analogs examined were based on the N-((o-phthalimidylalkyl)-iV-(a)-phenylalkyl)-amine (scaffold 66). An iterative process of testing. 

Irreversible inhibition occurs with organophos-phorus insecticides and chemical warfare agents (see p. 437) which combine covalently with the active site of acetylcholinesterase recovery of cholinesterase activity depends on the formation of new enzyme. Covalent binding of aspirin to cyclo-oxygenase... 

Knowledge of the mechanisms of action of acetylcholinesterase and of the reaction of organophosphorous compounds with esterases led to the development of drugs useful in the treatment of this kind of intoxication. The active site of acetylcholinesterase consists of two subsites ... 

C12. Gohen, J. A., and Oosterbaum, R. A., The active site of acetylcholinesterase and related esterases and its reaetivity towards substrates and inhibitors. In Handbuch der experimentellen Pharmacologie. Vol. XV. Gholinesterase and anticholinesterase agents (G. B. Koelle, ed.). Springer-Verlag, Berlin and New York, 1963. 

The action of OP nerve agents on the nervous system results from their effects on enzymes, particularly esterases. The most notable of these esterases is acetylcholinesterase. The active site of acetylcholinesterase comprises a catalytic triad of serine, histidine and glutamic acid residues and other important features of the enzyme are a gorge connecting the active site to the surface of the protein and a peripheral anionic site (Bourne etal., 1995,1999 Sussman etal., 1991 Thompson and Richardson, 2004), The OPs phosphy-late1 the serine hydroxyl group in the active site of the enzyme. 

Figure 5.3 The classical model of the active site of acetylcholinesterase.

Inhibitors of acetylcholinesterase are used both as poisons and as drugs. Among the most important inhibitors of acetylcholinesterase are a class of compounds known as organo-plwsphutes. One of these is the nerve agent Sarin (isopropyl-methylfluorophosphate). Sarin forms a covalently bonded intermediate with the active site of acetylcholinesterase. Thus, it acts as an irreversible, noncompetitive inhibitor. 

In living beings, these pesticides bind irreversibly to the active site of acetylcholinesterase (AChE) - enzyme involved in the transmission of nerve impulse. Electrochemical biosensors for measurement of these pesticides are based all on the inhibition of AChE and the inhibition degree is proportional to the pesticide concentration. 

These agents phosphorylate the active site of acetylcholinesterase. Once the phosphorylation is stable, it is not reversible within the length of time that the enzyme is active (the inactivation extends for the life of the enzyme). (See Table above). 

Once ingested, the liver converts malathion to the toxic reactive compound, malaoxon, by replacing the sulfur with an oxygen. Malaoxon then binds to the active site of acetylcholinesterase and reacts to form the covalent intermediate. Unlike the complex formed between diisopropylfluorophos-phate and acetylcholinesterase, this initial acylenzyme intermediate is reversible. However, with time, the enzyme-inhibitor complex "ages" (dealkylation of the inhibitor and enzyme modification) to form an irreversible complex. 

FIG. 2, Schematic illustrating the interaction of acetj Icholine (I), the carbamate carbaryl (II), and the organophosphate ehiorpynfos-oxon (IJI) with the active site of acetylcholinesterase (ACbE). The general rate of bound AChE hydrolysis is ACh > > carbaryl > chlorpyrifos-oxon. 

The carbamyl ester inhibitors compete with acetylcholine for the active site of the enzyme. Acetylcholinesterase becomes carbamylated as it cleaves the ester linkage. The carbamyl group prevents acetylcholine from binding to the active site of acetylcholinesterase for a period of minutes to hours. Upon decarbamylation, the enzyme regains its ability to cleave acetylcholine. 

Edrophonium, a competitive anticholinesterase, contains a quaternary ammonium which is attracted to an anionic pocket near the active site of acetylcholinesterase. Edrophonium does not act as long as... 

The organophosphorus inhibitors have very high affinity for the active site of acetylcholinesterase. Once bound, these agents inactivate the enzyme by phosphorylation. Regeneration of the enzyme is so slow that a single dose of DFP inhibits acetylcholinesterase for over a week. 

Acetylcholinesterase is a serine esterase whose catalytic mechanism is similar to that of the serine proteases. As with chymotrypsin and trypsin, the active site of acetylcholinesterase has serine as part of a Ser-His-Asp catalytic triad. The mechanism -will involve covalent tetrahedral and acyl enzyme intermediates in which the substrate is bonded covalently to the active-site Ser. The reaction starts with nucleophilic attack on... 

Fish rapidly absorb, metabolize, and excrete chlorpyrifos from the diet. The mechanism of action of chlorpyrifos occurs via phosphorylation of the active site of acetylcholinesterase after initial formation of chlorpyrifos oxon by oxidative desulfuration. In studies with chaimel catfish (Ictalurus punctatus), the oral hioavailability of chlorpyrifos was 41%, substantially higher than in mammals. Catfish muscle contained less than 5% of the oral dose with an elimination half-life (Tbl/2) of 3.3 days. Chlorpyrifos residues in whole catfish were more than 95% chlorpyrifos, while bile and urine primarily contained metabolites. The dephosphorylated metabolite trichloropy-ridinol (TCP) was the major metabolite in the blood while the glucuronide conjugate of TCP was the major metabolite in mine and bile. The toxic metabolite, chlorpyrifos oxon, was not detected in blood, tissues, or excreta. Extensive metabolism resulted in a low potential for chlorpyrifos to accumulate in catfish from dietary exposure. In both fish and mammals, TCP is a major biotransformation product. Chaimel catfish rapidly distribute waterborne chlorpyrifos into the blood and more slowly to peripheral tissues, with concentrations highest in fat and lowest in muscle. As was true with dietary chlorpyrifos, TCP was the major metabolite in blood and the glucuronide conjugate of TCP was the major metabolite in urine and bile. Pharmacokinetics and metabolism of waterborne chlorpyrifos in channel catfish were similar to the disposition of chlorpyrifos in other vertebrates. 

---