Structure of the Esteratic Site

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

like all animal esterases known so far, do not contain any specific prosthetic group. Upon hydrolysis, either by acids or by proteolytic enzymes (35), only amino acids or oligopeptides can be isolated. The enzymic activity decreases considerably during the degradation process (36), indicating that a complex structural arrangement is responsible for the catalytic effect. Therefore, the structure of the esteratic site can be derived only indirectly, i.e., by studies on the complete enzyme. 

In this respect, the most important information has been obtained from the effect of pH changes on enzymic activity. In Fig. 2 the pH-activity curves are represented for true cholinesterase (from Torpedo marmorata) and pseudo-ChE (from human serum), with ACh as substrate. The two curves are not only similar to each other, but also to the curves, characteristic for other, unspecific esterases (37). For the correct interpretation of such curves, it is important to make sure that only the protein in the 

Only very few among the common amino acids possess a pK within the range 5.8-7.0. Therefore, the imidazole ring of histidine was suspected very early to represent the group responsible for nucleophilic attack on the substrate (38). The pK of free imidazol is 6.9 (39) that of imidazol, contained in histidine or its peptides, varies between 5.6 and 7.1 (40). Imidazol is well known to form unstable acyl derivatives, which undergo spontaneous hydrolysis because of the presence of the resonating triad unit —-N—C= N— (41). In addition, imidazol and its derivatives catalyze the hydrolysis of certain esters, especially those derived from phenols (42). Likewise, the behavior of imidazol towards thio esters reflects exactly the specificity of ChE s (see IV, 4). Thus, thiol esters are split (43), whereas thiono esters are resistant (21). 

If one accepts the imidazol hypothesis, it becomes possible to formulate a more detailed mechanism of enzymatic hydrolysis, as shown in scheme E  

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The mechanism of action of the cholinesterases is much better undmstood than the mechanism of action of cholinoreceptors. The main function of cholinesterase i.e. the hydrolysis of ACh. is carried out by its esteratic site. In the structure of the esteratic site the hydroxyl of the amino-acid serine plays a central role When the ACh molecule attacks the active surface of cholinesterase the first event is the sorption of the trimethylammonium end of ACh on the anionic site of ChE. This sorption fixes the ACh molecule at the necessary distance from the esteratic site... 

The hydrophobic area surrounding the anionic site plays a more important role for butyrylcholinesterase than for acetylcholinesterase. The greater importance of this hydrophobic area for butyrylcholinesterase could help to explain and resolve some of the opposing views of earlier workers (A26). Kabachnik et al. (Kl) also proposed that in the vicinity of the esteratic site of butyrylcholinesterase there are two hydrophobic areas separated by a hydrophilic group. Differences in length and structure of the hydrophobic areas of the active surfaces of butyryl- and... 

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. 

A fruitful approach to this problem was based on the study of the inhibitory effect of quaternary ammonium ions, such as tetraethylammonium, as a function of pH (56). This type of inhibitors can combine only with the anionic sites, but does not possess any specific binding forces for the esteratic site. Therefore, any change in the inhibitory activity, when the pH is varied, must be ascribed to structural changes of the anionic sites, abolishing their charges. 

The detailed mechanism by which AChE and BChE hydrolyze ACh has been the subject of much research, especially since the crystal structure of the Torpedo califomica AChE was elucidated by Sussman et al. in 1991 [12]. (Reviews of these enzymes and their interactions can be found in Refs. [5,13]). This mechanism will be described here only briefly, as an introduction to the reaction of the enzyme with carbamates. The active site of AChE is located at the bottom of a 20 A-deep gorge, where acetylcholine fits in by attachment of the quaternary ammonium group to the so-called anionic site (mainly through cation interaction with the n electrons of Trp84), and by dipole interactions between the ester group and Ser200 at the esteratic site . 

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... 

Figure 1. Schematic of the probable physical structure of acetylcholinesterase. One physical region carries the esteratic site, which is proximal to one anionic site (Site I) the other region would carry at least four anionic sites, and would be homologous to the acetylcholine receptor of the motor end plate excitable membrane. Sites I and II are masked by DPA, but Site II can be regenerated at alkaline pH. Decamethonium (C10) would interact at least with Sites I and II whereas curare would bind at III and TV, and perhaps at II and III. Most quaternary salt substituents bind on the anionic chain [exo-binding (26, 36, 42.

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