Acetylcholinesterase serine residue

Most enzymes bind their substrates in a non-covalent manner but, for those that do bind covalently, the intermediate must be less stable than either substrate or product. Many of the enzymes that involve covalent catalysis are hydrolytic enzymes these include proteases, lipases, phosphatases and also acetylcholinesterase. Some of these enzymes possess a serine residue in the active site, which reacts with the substrate to form an acylenzyme intermediate that is attacked by water to complete the hydrolysis (Fignre 3.3). 

Tlie neurotransmitter acetylcholine is both a quaternary ammonium compound (see Box 6.7) and an ester. After interaction with its receptor, acetylcholine is normally degraded by hydrolysis in a reaction catalysed by the enzyme acetylcholinesterase. This enzyme contains a serine residue that acts as the nucleophile, hydrolysing the ester linkage in acetylcholine (see Box 13.4). This effectively acetylates the serine hydroxyl, and is an example of transesterification (see Section 7.9.1). For continuation of acetylcholine degradation, the original form of the enzyme must be regenerated by a further ester hydrolysis reaction. 

As a result, the penicillin occupies the active site of the enzyme, and becomes bound via the active-site serine residue. This binding causes irreversible enzyme inhibition, and stops cell-wall biosynthesis. Growing cells are killed due to rupture of the cell membrane and loss of cellular contents. The binding reaction between penicillinbinding proteins and penicillins is chemically analogous to the action of P-lactamases (see Boxes 7.20 and 13.5) however, in the latter case, penicilloic acid is subsequently released from the P-lactamase, and the enzyme can continue to function. Inhibitors of acetylcholinesterase (see Box 7.26) also bind irreversibly to the enzyme through a serine hydroxyl. 

Substances that block the serine residue in the active center of acetylcholinesterase [2j—e.g., the neurotoxin E605 and other organophosphates—prevent ACh degradation and thus cause prolonged stimulation of the postsynaptic cell. This impairs nerve conduction and muscle contraction. Curare, a paralyzing arrow-poison used by South American Indians, competitively inhibits binding of ACh to its receptor. 

The answer is D. Organophosphates react with the active site serine residue of hydrolases such as acetylcholinesterase and form a stable phosphoester modification of that serine that inactivates the enzyme toward substrate. Inhibition of acetylcholinesterase causes overstimulation of the end organs regulated by those nerves. The symptoms manifested by this patient reflect such neurologic effects resulting from the inhalation or skin absorption of the pesticide diazinon. 

It is important to know that the inhibition of acetylcholinesterase by OPs is through an attack on the relatively positive phosphorus atom by the hydroxyl group of a serine residue at the enzyme s site of action. Electron withdrawing substitutions within the OP tend to make the phosphorus more positive and, therefore, more reactive. Unfortunately, this type of substitution also makes the compound less stable hydrolytically. The discovery and development of OP insecticides has always been a balance between activity against the enzyme of the insect, selectivity in comparison with mammalian systems and stability within the insect. The binding of OPs to acetylcholinesterase is often irreversible. Typical OP insecticides are shown in Figure 3.3. 

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 ... 

The serine proteases act by forming and hydrolyzing an ester on a serine residue. This was initially established using the nerve gas diisopropyl fluorophosphate, which inactivates serine proteases as well as acetylcholinesterase. It is a very potent inhibitor (it essentially binds in a 1 1 stoichiometry and thus can be used to titrate the active sites) and is extremely toxic in even low amounts. Careful acid or enzymatic hydrolysis (see Section 9.3.6.) of the inactivated enzyme yielded O-phosphoserine, and the serine was identified as residue 195 in the sequence. Chy-motrypsin acts on the compound cinnamoylimidazole, producing an acyl intermediate called cinnamoyl-enzyme which hydrolyzes slowly. This fact was exploited in an active-site titration (see Section 9.2.5.). Cinnamoyl-CT features a spectrum similar to that of the model compound O-cinnamoylserine, on denaturation of the enzyme in urea the spectrum was identical to that of O-acetylserine. Serine proteases act on both esters and amides. 

Trimethylammonium trifluoroacetophenone (19) was found to be a highly effective inhibitor of acetylcholinesterase 37 f The ketone activated by an electron-withdrawing trifluoroacetyl group will enhance the tendency to add a nucleophile (the hydroxyl group of the catalytic serine residue of acetylcholinesterase) to form a tetrahedral adduct as an aldehyde inhibitor. 

Phosphylation of the OH moiety of the serine residue, being part of the catalytic triad in the esteratic center of acetylcholinesterase (AChE), represents the pathophysio-logically most important reaction resulting in enzyme deactivation. Inhibition of AChE was proven to be the predominant major reaction in vivo that causes death... 

Serine residues occurring in the active sites of serine proteases (e.g., chymotryp-sin) or other hydrolyases (e.g., acetylcholinesterase) are specifically modified by diisopropylfluorophosphate in a reaction that is essentially irreversible. 

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... 

Acetylcholine is a neurotransmitter that relays nerve impulses across the neuromuscular Junction. Acetylcholinesterase (AcChE) rapidly breaks dovm acetylcholine, thereby loweringits concentration in the synaptic cleft and ensuring that nerve impulses are of a finite length. As shown in Fig. 17.38, a nucleophilic serine residue reacts with the substrate to form an acetyl-serine intermediate (100) with concomitant release of choline. This intermediate is then rapidly hydrolyzed by wa-... 

While A-esterase(s) and B-esterases interact kinet-ically with paraoxon in a similar fashion (Figure 3), the molecular events occurring at their active sites during catalysis are probably very different. The active site of B-esterases such as acetylcholinesterase has been well characterized and contains a serine residue that is phosphorylated by paraoxon at the hydroxyl group. In contrast, the active site of A-est-erase(s) has not been studied as extensively, but it likely does not contain a serine residue that participates in the hydrolysis of paraoxon. Additionally, A-esterase(s) requires a divalent cation like calcium for activity, whereas B-esterases do not. 

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). 

These organic phosphates inhibit acetylcholinesterase by reacting with the active-site serine residue to form a stable phosphorylated derivative. They cause respiratory paralysis by blocking synaptic transmission at cholinergic synapses. 

Acetylcholinesterase. Altered acetylcholinesterase less sensitive to organophosphorus and carbamate insecticides has been observed in a wide variety of insects and mites (51). Acetylcholinesterase inhibiting insecticides phosphorylate or carbamylate the serine residue in the active site of the enzyme preventing vital catalysis of acetylcholine. Resistance due to reduced sensitivity to inhibition of this target enzyme has been found in house fly, mosquitoes, green rice leafhopper, and both phytophagous and predacious species of mites. 

Some useful relationships can then be derived, e.g. k = ki/Kn (Main and Iverson, 1966). In addition, k, = In 2//50 (Aldridge, 1950) which allows easy estimation of, (The I50 is the concentration of inhibitor, which inhibits the enzyme by 50%). These constants have been measured for many OP chemical warfare agents and also pesticides (e.g. Gray and Dawson, 1987). The hydrolysis reaction for acetylated acetylcholinesterase is fast (Koelle, 1992), in the region of 100 ps (Lawler, 1961 O Brien, 1976). The key to the powerful anticholinesterase effects of OPs is what happens after inhibition by these compounds. In the case of OPs, hydrolysis of the phosphylated serine residue is much slower2 than the acetylated analogue. 

Over 80 different (3-lactamases are now known. One classification is a system that divides the enzymes into three classes A, B, and C. Classes A and C are active-site serine enzymes. The serine residue in class A enzymes is at position 70. This class contains four major (3-lactamases 749/C (from B. licheniformis), PCI (from S. aureus), 569/H P-lactamase I (from B. cereus), and PBR322 and RTEM (from E. coli). As with other serine-type hydrolytic enzymes (acetylcholinesterase, trypsin), the mechanism of action requires initial formation of an acylated enzyme, in this case acylation of ser-70 followed by hydrolysis of the derivative to regenerate the enzyme ... 

Acetylcholinesterase works through a serine residue that forms a covalent intermediate with acetylcholine. The intermediate is then hydrolyzed to give free enzyme, acetate, and choline. 

Acetylcholinesterase (AchE) hydrolyses the neurotransmitter acetylcholine and yields acetic acid and choline. AchE is a serine hydrolase inhibited by organophosphorus poisons, as well as by carbamates and sulfonyl halides which form a covalent bond to a serine residue in the active site. AchE inhibitors are used in the treatment of various disorders. ... 

Acetylcholinesterase is inhibited by organic fluorophosphates through the formation of covalent phosphate esters with the serine residue at the active site of the enzyme. The reactivation of the enzyme can be achieved by treatment with hydroxylamine which acts as a transesterification and not an aminolysis agent (119). The transition state symbiosis may be the determinant for the specificity. 

Compounds which appear to be somewhat more effective antidotes have now been evolved. They act by releasing the phosphoryl group which has become attached to the serine residue in the inhibited enzyme. For example, DFP reacts with acetylcholinesterase (and certain other enzymes) to produce a phosphoryl-bonded enzyme which becomes inactive or inhibited (12.151). 

Acetylcholine is a neurotransmitter at cholinergic sites and acetylcholinesterase is the esterase, which brings about the hydrolysis of acetylcholine after it has performed its purpose of neurotransmission at synapses and cholinergic effector sites. Acetylcholine binds to acetylcholinesterase at two sites, namely the anionic site and the esteratic site. The quaternary nitrogen of choline forms an electrostatic link at the anionic site, while the carbonyl group binds to a serine residue at the esteratic site. The reaction for acetylcholine can be visualized as shown in eqn (10.1) below, E being the enzyme, AX acetylcholine, EAX is a reversible Michaelis-Menten complex and A is acetate ... 

Carbamates (urethanes). From 1947 onwards, carbamate insecticides have been brought very much to the fore on the grounds that they are less toxic to humans and more selective among insect species. These substances act by acylating serine residues in acetylcholinesterase (the R N CO-group is transferred to the hydroxyl-group of serine, thus forming a... 

FIGURE 5.46 Interaction of the serine hydroxyl residue in the catalytically active site of acetylcholinesterase enzyme with esters of organophosphates or carbamates. The interaction leads to binding of the chemical with the enzyme, inhibition of the enzyme, inhibition of acetylcholine hydrolysis, and thus accumulation of acetylcholine in the synapses. 

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. 

Carbamate Insecticides. Carbamate insecticides interact with acetylcholinesterase in exactly the same way as OPs, with the hydroxyl group in the serine at the enzyme s active site attacking the carbamate residue in the insecticide. However, the binding to the active site is reversible. Typical carbamate insecticides are shown in Figure 3.4. 

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