Serine residue cholinesterases

The inhibition of brain cholinesterase is a biomarker assay for organophosphorous (OP) and carbamate insecticides (Chapter 10, Section 10.2.4). OPs inhibit the enzyme by forming covalent bonds with a serine residue at the active center. Inhibition is, at best, slowly reversible. The degree of toxic effect depends upon the extent of cholinesterase inhibition caused by one or more OP and/or carbamate insecticides. In the case of OPs administered to vertebrates, a typical scenario is as follows sublethal symptoms begin to appear at 40-50% inhibition of cholinesterase, lethal toxicity above 70% inhibition. 

Organophosphate and carbamate pesticides are potent inhibitors of the enzyme cholinesterase. The inhibition of cholinesterase activity by the pesticide leads to the formation of stable covalent intermediates such as phosphoryl-enzyme complexes, which makes the hydrolysis of the substrate very slow. Both organophosphorus and carbamate pesticides can react with AChE in the same manner because the acetylation of the serine residue at the catalytic center is analogous to phosphorylation and carbamylation. Carbamated enzyme can restore its catalytic activity more rapidly than phosphorylated enzyme [17,42], Kok and Hasirci [43] reported that the total anti-cholinesterase activity of binary pesticide mixtures was lower than the sum of the individual inhibition values. 

Another different class of inhibitors binds covalently to specific amino acids in the enzyme and these are referred to as irreversible inhibitors. The organophosphorus compounds, of which nerve gases are examples, inactivate enzymes which rely on the hydroxyl group of serine residues for their activity, e.g. cholinesterase (EC 3.1.1.8). 

Other serine hydrolases such as cholinesterases, carboxylesterases, lipases, and fl-lactamases of classes A, C, and D have a hydrolytic mechanism similar to that of serine peptidases [25-27], The catalytic mechanism also involves an acylation and a deacylation step at a serine residue in the active center (see Fig. 3.3). All serine hydrolases have in common that they are inhibited by covalent attachment of diisopropyl phosphorofluoridate (3.2) to the catalytic serine residue. The catalytic site of esterases and lipases has been less extensively investigated than that of serine peptidases, but much evidence has accumulated that they also contain a catalytic triad composed of serine, histidine, and aspartate or glutamate (Table 3.1). 

Based on the above discussion it was thought that the trifluoro-methyl ketones would be more polarized and thus create a great electrophilicity on the carbonyl carbon which facilitates -OH attack by the serine residue. Yet there is no carbon-oxygen bond to be cleaved In the ketone moiety, and therefore the enzyme-trifluoromethyl ketone transition state complex does not undergo catalytic conversion. The above rationale seems reasonable as trifluoromethyl ketones were found to be extraordinary selective and potent inhibitors of cholinesterases (56) of JHE from T. ni (57) and of meperidine carboxylesterases from mouse and human livers (58). Since JH homologs are alpha-beta unsaturated esters, a sulfide bond was placed beta to the carbonyl in hopes that it would mimic the 2,3-olefln of JHs and yield more powerful inhibitors (54). This empirical approach was extremely successful since it resulted in compounds that were extremely potent inhibitors of JHEs from different species (51,54,59). 

Cholinesterase.s that have been cxpo.sed to phosphorylat-ing agents (e.g.. sarin) become refractory to reactivation by cholinesterase reactivators. The process is called ttging and occurs both in vivo and in vitro with AChE and BuChE. Aging occurs by partial hydrolysis of the phosphorylated nioicty that is attached to the serine residue at the estcratic rite of the enzyme (Fig. 17-17). 

The first step of the reaction of cholinesterase with the OP.v is similar to its interaction with ACh as its catalytic serine residue is phosphorylated and the acid component is released from the educt (Figure 9.2). This phosphorylation is nearly irreversible and the cholinesterase is no longer able to cleave ACh, causing a continuous stimulus. This uncontrolled reaction results in an intoxication. ... 

Carboxypeptidase A (approx. 35 kDa MW, Sigma Chemical) functions both as a peptidase and an esterase it is in this latter mode that it can serve as a detector for cholinesterase inhibitors. Unlike the other enzymes such as AChE or BChE, it does not have a serine residue in the active site. TPPSi forms a complex with the enzyme and, upon challenge with the cholinesterase inhibitor eserine (physostigmine) in water, exhibits a change in the absorbance spectrum with a new peak and a marked increase in absorbance at 423 nm. This suggests TPPS, may not be completely displaced from the active site. For actual sensor operations, the use of an enzyme such as BChE or carboxypeptidase in place of (or in addition to) AChE will allow for potential identification of the analyte based on different specificities/sensitivities of the enzyme. Enzymes such as OPH, which are not readily available, may be difficult to obtain in large quantities the supply of AChE is often limited perhaps due to the capture of electric eels, while proteins such as BChE (from horse blood) and carboxypeptidase (pancreas) are more readily available from slaughterhouses. 

Both hydroxylamine and PAM act by releasing the phosphoryl group which has become attached to the serine residue of the inhibited enzyme. Other cholinesterase inactivators, generally more effective than PAM, but not equally effective against all gases are obidoxime and HI-6 (12.153). Compounds for pre-treatment therapy are under investigation [84,85]. 

An important feature of enzymes is that their active sites can often be occupied by, or react with, molecules other than the substrate, leading to inhibition of enzyme activity. Several inhibition mechanisms are known, but it is necessary only to distinguish between irreversible and reversible inhibition. Irreversible inhibition arises when the inhibitor molecule I dissociates very slowly or not at all from the enzyme active site. The best-known examples occur when I reacts covalently with a critical residue in the active site. Inhibition of cholinesterase enzymes by the reaction of organo-phosphorus compounds with a serine residue is a case in point. This type of inhibition is said to be noncompetitive—enzyme activity cannot be restored by addition of excess substrate. So although addition of I reduces V,n x, Km is unaffected. The double-reciprocal plot in such cases has the same. v-axis intercept as the plot for the uninhibited enzyme, but greater slope. 

Figure 17-16 Phosphorylation and reactivation of cholinesterase. A. Phosphorylation of serine by isofluorphate. B. Phosphorylated serine at the esteratie site. C Nucleophilic attack on phosphorylated residue by 2-PAM. D. Free enzyme.

The human lysosmal serine carboxypeptidase CatA is a member of the a/p hydrolase enzyme family and therefore shares structural homology with yeast carboxypeptidase Y and cholinesterases [4] (Figure 23.1). The common feature shared by members of this family is a three-dimensional structure of eight p sheets connected by a-heUces resulting in a typical topological localization of the catalytic triad, in which only the His residue is conserved [2]. 

DFP is able to bind to many proteins with serine, tyrosine, and other residues therefore, it is an inhibitor of proteases and other esterases different from cholinesterases. This capacity has triggered interest in using it for toxicological, pharmacological, and biomedical research. As a consequence, DFP is accessible and has been used in several fields as follows ... 

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