Cholinesterases esteratic site

Figure 6.1 Synthesis and metabolism of acetylcholine. Choline is acetylated by reacting with acetyl-CoA in the presence of choline acetyltransferase to form acetylcholine (1). The acetylcholine binds to the anionic site of cholinesterase and reacts with the hydroxy group of serine on the esteratic site of the enzyme (2). The cholinesterase thus becomes acetylated and choline splits off to be taken back into the nerve terminal for further ACh synthesis (3). The acetylated enzyme is then rapidly hydrolised back to its active state with the formation of acetic acid (4)...

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... 

Most cholinesterase inhibitors inhibit the enz)nne by acylating the esteratic site on the enzyme surface. Physostigmine and neostigmine are examples of... 

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-... 

As given in classification, these agents are of two type e.g. reversible and irreversible. The reversible anticholinesterases have a structural resemblance to acetylcholine, are capable of combining with anionic and esteratic sites of cholinesterase as well as with acetylcholine receptor. The complex formed with the esteratic site of cholinesterase is less readily hydrolyzed than the acetyl esteratic site complex formed with acetylcholine. Edrophonium forms reversible complex with the anionic site and has shorter duration of action. Also, neostigmine and edrophonium have a direct stimulating action at cholinergic sites. 

Irreversible cholinesterases are mostly organophosphorus compounds and combine only with esteratic site of cholinesterase and that site gets phosphorylated. The hydrolysis of phosphorylated site produces irreversible inhibition of cholinesterase. And, because, of this property, the therapeutic usefulness is very limited. Most of the compounds are used as insecticides e.g. parathion, malathion and war gases e.g. tabun, sarin, soman etc. 

VI. Structure of the Esteratic Site 1. pH Activity Curves of Cholinesterases... 

The second pK, 8.5-9.5, derived from the pH-activity curves, is much more difficult to interpret. This pK is naturally absent in the system imidazol + ester (21). It is also subject to much greater variation than pK0. This has been demonstrated for a variety of substrates (Fig. 3), but is especially prominent when thiol esters are being studied (Figs. 4 and 5). In the system eel esterase-acetylthiocholine, no decrease of activity is observed on the alkaline side up to pH 11, and for plasma cholinesterase-acetylthiocholine the decrease is very much delayed, when compared with the oxy ester, acetylcholine (see Fig. 2). Similar observations have been made with other esterases and other thiol esters (44)- They indicate that the second component 02 of the esteratic site, to which pK has to be ascribed, may be less essential for certain substrates than for others. 

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... 

Acyl Cholinesterases. Acetylcholinesterase (AChE EC 3.1.1.7 CAS 9000-81-1) is the serine esterase which catalyzes the hydrolysis of acetylcholine and possesses an esteratic site, and which is responsible for unspecific hydrolyses of several substrates. Also, butyrylcholinesterase (EC 3.1.1.8 CAS 9001-08-5) has been sometimes used for asymmetric hydrolysis of esters. Acetylcholinesterase has been used for... 

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... 

The enzyme cholinesterase, effecting the degradation of ACh, consists of two active sites, the anionic and the esteratic site (Figure 9.1). In the first step, ACh is attached to the anionic site and the acetyl group of the ACh molecule is transferred to the amino acid serine. The resulting serine ester is then cleaved hydrolytically and the enzyme is regenerated. ... 

Cholinesterase is now considered to react with substrates and competitive inhibitors by the initial formation of an enzyme affinity complex which enables the serine hydroxyl group at the esteratic site to become acylated. The acylated group can then react with any nucleophilic reagent to regenerate the free enzyme. The overall scheme is as follows ... 

The basis of our current understanding of the two types of active site which are present in cholinesterase has been provided by Wilson and Bergmann (W30), who substantiated the concept of anionic and esteratic sites introduced by Zeller and Bisegger (Z2). Although it is now generally accepted that the hydrolysis of the ester bond of the substrate occurs at the esteratic site, there is still some uncertainty regarding the existence of the anionic site in butyrylcholinesterase an alternative site has been proposed by Augustinsson (A28). 

The discovery that organophosphates such as diisopropyl fluoro-phosphate (DFP) inhibit cholinesterase by irreversible phosphorylation of a basic group at the esteratic site led to the use of P P]DFP to ascertain the chemical nature of the DFP-binding site. Jansz et al. (J2) found that the structure of the P peptide of horse serum cholinesterase was Phe-Glu-Ser-Ala-Gly-Ala-Ala-Ser This indicated the serine hydroxyl as the... 

It has already been mentioned that there are some doubts A26) about the existence of an anionic site in human or horse cholinesterase. Comparative kinetic studies using a series of pyridylcarbinol acetates as substrates have shown that acetylcholinesterase from T. marmorata electric organ and the plasma cholinesterases from horse and man have similar esteratic sites. It was also shown that the electric eel organ enzyme has an anionic site, whereas the second site of butyrylcholine... 

Comprehensive reviews (Kl, Ul) of the active sites of cholinesterase both postulated the presence not only of an esteratic site for butyrylcholinesterase but also of an anionic site. Additionally, in the region of the anionic site, there are two hydrophobic areas, one directly surrounding the anionic group and the second located at some distance from it (Kl). The presence of hydrophobic areas has been established (B32, C3, H29, H45, MIO) by the use of fluorescent probes with spectral responses which reflect the environment of the probe. Such probes can be used to monitor changes in the conformations of enzymes and can be designed to be active-site-directed, competitive inhibitors (H30). Aspects of the spectroscopy of intrinsic and extrinsic fluorescent probes have been reported (C3). 

Thus, it is probable that the active site of cholinesterase comprises anionic and esteratic subsites and also, as with most proteins, hydro-phobic areas. The anionic site determines specificity with respect to the choline moiety, while the actual catalytic process takes place at the esteratic site (F8). 

The quaternary ammonium compound Tris [tris-(hydroxymethyl)-aminomethane] has been found to be a competitive inhibitor of horse plasma cholinesterase using butyrylcholine as substrate (P5), with inhibition occurring at concentrations commonly used in buffer solutions. Tris is believed to compete for the esteratic site but, in the absence of Mg and Ca, the enzyme was activated. The exact role of the cations was not investigated, but a complex of the cation with an amino group could be the effective inhibitor. Similar results were obtained using acetylcholinesterase from r. marmorata. 

Irreversible inhibitors are effectively esteratic site inhibitors which, like true substrates, react with the hydroxyl group of serine at the catalytic active site. Such inhibitors, sometimes referred to as acid-transferring inhibitors, include the organophosphates, the organo-sulfonates, and the carbamates. All form acyl-enzyme complexes which, unlike substrate-enzyme intermediates, are relatively stable to hydrolysis. Indeed, the phosphorylated enzyme intermediates have half-lives from a few hours to several days (A12), whereas the sulfonated or carbamylated enzyme complexes have much shorter half-lives—several minutes to a few hours. Several strong lines of direct evidence point to the formation of an acyl complex—the isolation of phosphorylated serine from hydrolysates of horse cholinesterase (J2), complex formation and carbamylation (02), and the sulfonation of butyrylcholinesterase by methanesulfonyl fluoride in the presence of tubocurarine and eserine (P6). 

Carbamates generally act quickly. They are strongly toxic to a wide range of insect pests, but have a weak effect on the red spider mite. Some of them exhibit systemic characteristics. The duration of their action varies considerably. In a similar manner to the phosporic acid esters discussed later, they exert their action by paralysing the cholinesterase enzyme. During this process, the carbamate part of the molecule is attached to the esteratic site, and the aromatic part to the anionic site of the cholinesterase enzyme. As the distance between the esteratic and anionic sites is SO nm in the cholinesterase molecule, carbamate insecticides will be most efficient if the distance between the two groups to be bound to the two sites of the enzyme is also 50 nm. (Metcalf and Fukuto, 1965 1967 Fukuto et ai, 1967). 

CHOLINESTERASE REACTIVATORS Although the phosphorylated esteratic site of AChE undergoes hydrolytic regeneration at a slow or negligible rate, nucleophilic agents, such as hydroxylamine (NH OH), hydroxamic adds (RCONH-OH), and oximes (RCH=NOH), reactivate the enzyme more rapidly than does spontaneous hydrolysis. Reactivation with prahdoxime (Figure 8-1E) occurs at a million times the rate of that with hydroxylamine. Several h/s-quaternary oximes are even more potent as reactivators for insectidde and nerve gas poisoning (e.g., HI-6, used in Europe as an antidote). 

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