Cholinesterases anionic 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... 

It may be noted that many of the anticholinesterases of the competitive type are equally potent as inhibitors of a- and / -cholinesterases. We should add, however, that the existence of an anionic site in / -cholinesterases has been questioned.1... 

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. 

The Number of Anionic Sites in True and Pseudo-Cholinesterase. 151... 

Fio. 10. The arrangement of the cationic inhibitor tetrametliylammonium at the anionic site of cholinesterases. (For true ChE, the arrangement of lowest potential energy is shown, with the centers of the three ions involved lying on a straight line.)... 

On the Forces Acting Between Quaternary Ammonium Ions and the Anionic Sites of Cholinesterases... 

Free Energies and Interionic Distances for the Association of Ammonium Ion with the Anionic Sites of Cholinesterases Calculations are based on Equation (10)... 

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... 

A series of novel l, 3-dihydroxyxanthone Mannich base derivatives were synthesized, structure elucidated, and evaluated for anticholinesterase activity. Most of the target compounds exhibited moderate to good inhibitory activities with the IC50 values at micromole level concentration against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Among them, 2 - ((diethylamino )methyl) -1 -hydroxy- 3 - (3 -methylbut-2-enyloxy) -9H-xanthen-9-one showed potent inhibitory activity against AChE and the best inhibitory activity against BuChE. The Mannich base derivatives were likely to bind to the active site (AS) and the peripheral anionic site (PAS) of cholinesterases [169]. 

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. ... 

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). 

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). 

Many drugs of therapeutic importance, e.g., hexafluorenium, pancuronium (S9, SIO), and succinylcholine itself, are bis quaternary ammonium complexes which are reversible inhibitors of cholinesterase. One of the charged groups occupies the anionic site, but there is uncertainty regarding the attachment of the second quaternary nitrogen group. Experimental evidence shows that the bis quaternary compoimds are more than twice as potent (mole for mole) as inhibitors of cholinesterase than are simple quaternary ammonium compounds (M4). 

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). 

---