Active site enzymes cholinesterases

Fluorescent Equilibrium Probes. Himel and co-workers (23, 24, 25) have synthesized active-site-directed fluorescent equihbrium probes which are competitive inhibitors of the active site of cholinesterase enzymes. The fluorescence intensity of the probe-enzyme complex is decreased by any foreign molecule (insecticide) which competes with the equilibrium fluorescent probe for the active site of the enzyme or which changes the equilibrium dynamics by exo area reaction with the enzyme. This highly specific and sensitive spectroscopic method is being developed as an analytical method for insecticides (26). 

Anticholinesterase insecticides phosphorylate the active site of cholinesterase in all parts of the body. Inhibition of this enzyme leads to accumulation of acetylcholine at affected receptors and results in widespread toxicity. Acetylcholine is the neurohormone responsible for physiologic transmission of nerve impulses from preganglionic and postganglionic neurons of the cholinergic (parasympathetic) nervous system, preganglionic adrenergic (sympathetic) neurons, the neuromuscular junction in skeletal muscles, and multiple nerve endings in the central nervous system (Fig. 10-5). 

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). 

FIGURE 14.11 The pH activity profiles of four different enzymes. Trypsin, an intestinal protease, has a slightly alkaline pH optimnm, whereas pepsin, a gastric protease, acts in the acidic confines of the stomach and has a pH optimmn near 2. Papain, a protease found in papaya, is relatively insensitive to pHs between 4 and 8. Cholinesterase activity is pH-sensitive below pH 7 but not between pH 7 and 10. The cholinesterase pH activity profile suggests that an ionizable group with a pK near 6 is essential to its activity. Might it be a histidine residue within the active 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)...

Fig. 11.2. Schematic representation of the primary structure of secreted AChE B of N. brasiliensis in comparison with that of Torpedo californica, for which the three-dimensional structure has been resolved. The residues in the catalytic triad (Ser-His-Glu) are depicted with an asterisk, and the position of cysteine residues and the predicted intramolecular disulphide bonding pattern common to cholinesterases is indicated. An insertion of 17 amino acids relative to the Torpedo sequence, which would predict a novel loop at the molecular surface, is marked with a black box. The 14 aromatic residues lining the active-site gorge of the Torpedo enzyme are illustrated. Identical residues in the nematode enzyme are indicated in plain text, conservative substitutions are boxed, and non-conservative substitutions are circled. The amino acid sequence of AChE C is 90% identical to AChE B, and differs only in the features illustrated in that Thr-70 is substituted by Ser.

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... 

Methyl parathion itself is not a strong cholinesterase inhibitor, but one of its metabolites, methyl paraoxon, is an active inhibitor. Methyl paraoxon inactivates cholinesterase by phosphorylation of the active site of the enzyme to form the dimethylphosphoryl enzyme. Over the following 24-48 hours there is a process, called aging, of conversion to the monomethylphosphoryl enzyme. ... 

Reversible cholinesterase inhibitors form a transition state complex with the enzyme, just as acetylcholine does. These compounds are in competition with acetylcholine in binding with the active sites of the enzyme. The chemical stracture of classic, reversible inhibitors physostigmine and neostigmine shows their similarity to acetylcholine. Edrophonium is also a reversible inhibitor. These compounds have a high affinity with the enzyme, and their inhibitory action is reversible. These inhibitors differ from acetylcholine in that they are not easily broken down by enzymes. Enzymes are reactivated much slower than it takes for subsequent hydrolysis of acetylcholine to happen. Therefore, the pharmacological effect caused by these compounds is reversible. 

Various esterases exist in mammalian tissues, hydrolyzing different types of esters. They have been classified as type A, B, or C on the basis of activity toward phosphate triesters. A-esterases, which include arylesterases, are not inhibited by phosphotriesters and will metabolize them by hydrolysis. Paraoxonase is a type A esterase (an organophosphatase). B-esterases are inhibited by paraoxon and have a serine group in the active site (see chap. 7). Within this group are carboxylesterases, cholinesterases, and arylamidases. C-esterases are also not inhibited by paraoxon, and the preferred substrates are acetyl esters, hence these are acetylesterases. Carboxythioesters are also hydrolyzed by esterases. Other enzymes such as trypsin and chymotrypsin may also hydrolyze certain carboxyl esters. 

Inhibition of the cholinesterase enzymes depends on blockade of the active site of the enzyme, specifically the site that binds the ester portion of acetylcholine (Fig. 7.48). The organophosphorus compound is thus a pseudosubstrate. However, in the case of some compounds such as the phosphorothionates (parathion and malathion, for example), metabolism is necessary to produce the inhibitor. 

Certain therapeutic effects can be attributed to the inhibition of specific enzymic reactions. The inhibition of cholinesterase (Section 1.06.3), orotidylate pyrophosphorylase (Section 1.06.5) and of dihydrofolate reductase (Section 1.06.6.3) have already been discussed. They illustrate two modes of action, chemical alteration of the enzyme and competition with a substrate for the active site. 

Earlier work in this field [28] indicated that acetylcholinesterase enzymes would be suitable biomolecules for the purpose of pesticide detection, however, it was found that the sensitivity of the method varied with the type and source of cholinesterase used. Therefore the initial thrust of this work was the development of a range of enzymes via selective mutations of the Drosophila melanogaster acetylcholinesterase Dm. AChE. For example mutations of the (Dm. AChE) were made by site-directed mutagenesis expressed within baculovirus [29]. The acetylcholinesterases were then purified by affinity chromatography [30]. Different strategies were used to obtain these mutants, namely (i) substitution of amino acids at positions found mutated in AChE from insects resistant to insecticide, (ii) mutations of amino acids at positions suggested by 3-D structural analysis of the active site,... 

The covalent bond between OP and the active site serine of intact cholinesterase is stable, but not irreversible. Hydrolysis can occur with a half-life of between 10 and 35,000 min, depending on the enzyme, OP, temperature, pH, and buffer composition. The adduct becomes irreversibly bound to the enzyme after one of the alkyl groups on the OP is lost in a step called aging (Benschop and Keijer, 1966 Michel et al., 1967). The dealkylated OP makes a stable salt bridge with the protonated histidine of the catalytic triad, so that histidine is no longer available for the dephosphorylation step that would otherwise have restored the enzyme to an uninhibited state. Hundreds of scientists have contributed to this understanding... 

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... 

It has been noted that certain substances slow down and even stop enzyme-controlled reactions they are called inhibitors. Competitive inhibition occurs when a molecule has a similar molecular configuration to the substrate it therefore competes with the normal substrate for the active site and may slow down the reaction. The degree of inhibition depends on the relative concentrations of the substrate and inhibitor. Non-competitive inhibition occurs when the inhibitor attaches itself permanently to the active site the extent of inhibition depends entirely on the inhibitor concentration and cannot be altered by changing the amount of substrate present. Arsenic and heavy metals such as mercury and silver are toxic because they are inhibitors (non-competitive). Nerve gas, developed during the Second World War, is another example of an inhibitor it combines competitively with the enzyme cholinesterase and slows down the transmission of nerve impulses from one cell to another. 

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