Esterase enzyme inhibition

Emphasis is given to the critical role of metabolism, both detoxication and activation, in determining toxicity. The principal enzymes involved are described, including monooxygenases, esterases, epoxide hydrolases, glutathione-5 -transferases, and glucuronyl transferases. Attention is given to the influence of enzyme induction and enzyme inhibition on toxicity. 

Some OP compounds induce delayed neurotoxic effects ("delayed neuropathy") after acute poisoning. This delayed neurotoxic action is independent of cholinesterase inhibition but related to phosphorylation of a specific esterasic enzyme in the nervous tissue, called "neurotoxic esterase" or "neuropathy target esterase" (NTE) (Johnson, 1982). NTE is present in the nervous tissue, liver lymphocytes, platelets, and other tissues, but its physiological function is unknown. There is a rather large inter-individual variation of lymphocyte and platelet NTE activity (Table 2). 

We must stress that organo-phosphorus compounds are not specific inhibitors for the cholinesterases, but are rather inhibitors for enzymes possessing carboxylic esterase activity. All the enzymes mentioned below will hydrolyse carboxylic esters. However, not all esterases are inhibited, for example, A-esterase which hydrolyses phenyl acetate is not inhibited by organo-phosphorus compounds. 

A classification based on the interaction of esterases with organophosphates (2.1) has been introduced by Aldridge [64] class A-esterases hydrolyze organophosphate esters while B-esterases are irreversibly inhibited by them. Another, lesser-used criterion, is the effect of sodium 4-(hydroxymer-curio)benzoate or Hg2+, which inhibits A-esterases but has little effect on B-esterases. Class C-esterases, enzymes that do not interact at all with organophosphates, have been added to the classification system [64][65],... 

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. 

Enzymatic techniques have also been employed in the analysis of these compounds. The toxicity of carbamate insecticides is due to the inhibition of the enzyme acetylcholine esterase, so the determination of these compounds can be achieved by enzyme inhibition (2,83,119), bioassay (118,167), or enzyme-linked immunosorbent assay (ELISA) (168-171). In the detection of carbamates by fluorimetric enzyme inhibition, the effluent from a reversed-phase chromatographic column was incubated with cholinesterase, which was introduced via a postcolumn reagent delivery pump. Then, the resulting partially inhibited cholinesterase was reacted with N-methyl indoyl acetate to produce a fluorophore and a reduction in the baseline fluorescence (172). 

AChEs, BuChEs, and CaEs are specialized carboxylic ester hydrolases classed among the B esterases, enzymes that are inhibited by OPs. Another class of enzymes are the A esterases (e.g., paraoxonase and DFPase) that actively hydrolyze OPs, destroying their toxic potential... 

OPs are known to induce time-delayed neurotoxicity. This is due to the inhibition of an esterase in nerve tissue, neuropathy target esterase (NTE), that is also found in muscle and blood cells. The NTE level in the blood is an indicator of the inhibition of the enzyme. Inhibition of NTE and aging, the process of following the OP binding to an active esterase site that prevents the reactivation of the site, is important for selection of an antidote against certain OP nerve agents. It is of primary concern for Novichok agent. There is little information available on OP-caused neurotoxicity and the cardiac toxicity. 

Standard therapy of OP poisoning consists of the administration of a combination of atropine, oxime, and diazepam with other supportive measures when necessary. However, the possibility of addition of purified enzymes such as AChE, ChE, CarbE, and A-esterases to this therapeutic scheme has been considered and preliminary experiments in animals have shown much better protective effect after addition of exogenous enzymes. In this respect, protective effects of AChE, ChE, and CarbE are based on formation of covalent conjugates or phosphory-lated enzymes in the stoichiometric ratio 1 1. Capacity for binding of these enzymes is limited by the number of active sites on the enzyme to which OP molecules can be bound. This means that more enzymes have to be administered in order to achieve better detoxification of OPs which may not always be possible due to adverse effects. This can also be infiuenced by differences in the extent of spontaneous reactivation of these enzymes inhibited by OP. 

P15. Porter, G. R., Rydon, H. N., and Schofield, J. A., Mechanism of esterase action Nature of the reactive serine residue in enzymes inhibited by organophosphorus compounds. Nature 182, 927 (1958). 

The previous discussion of amino acid catabolic disorders indicates that catabolic processes are just as important for the proper functioning of cells and organisms as are anabolic processes. This is no less true for molecules that act as neurotransmitters. To maintain precise information transfer, neurotransmitters are usually quickly degraded or removed from the synaptic cleft. An extreme example of enzyme inhibition illustrates the importance of neurotransmitter degradation. Recall that acetylcholine is the neurotransmitter that initiates muscle contraction. Shortly afterwards, the action of acetylcholine is terminated by the enzyme acetylcholinesterase. (Acetylcholine must be destroyed rapidly so that muscle can relax before the next contraction.) Acetylcholinesterase is a serine esterase that hydrolyzes acetylcholine to acetate and choline. Serine esterases have catalytic mechanisms similar to those of the serine proteases (Section 6.4). Both types of enzymes are irreversibly inhibited by DFP (diisopropylfluorophosphate). Exposure to DFP causes muscle paralysis because acetylcholinesterase is irreversibly inhibited. With each nerve impulse, more acetylcholine molecules enter the neuromuscular synaptic cleft. The accumulating acetylcholine molecules repetitively bind to acetylcholine receptors. The overstimulated muscle cells soon become paralyzed (nonfunctional). Affected individuals suffocate because of paralyzed respiratory muscles. 

To develop a PD model for cholinesterase-inhibiting compounds, the steady-state levels (p,mol) of B-esterase enzymes (AChE, BuChE. and CarbE) were determined for the variou.s tissues (c.g., brain, blood, liver, and diaphragm) based on the rates of enzyme synthesis (zero-order) and degradation (first-order) (Gearhart et al., 1990). The... 

Various anticholinestera.scs are biologically potent chemicals that inhibit choIinesCera.scs via covalent interaction at the serine residue of the active site. Although enzyme inhibition may be reversible, there is the potential that other proteins may be modified and that several related enzymes may be inhibited. In many cases, it cannot be determined if the immunomodulatory effects are the result of inhibition of esterases or due to related mechanlsnis. 

In vitro, hundreds of proteins have been shown (o bind OPs covalently. Representative examples arc listed in Table 3, All serine esterases, serine peptidases, and serine proteases bind OPs covalently, resulting in enzyme inhibition. The site of OP binding is the serine residue in the catalytic site. There is at least one example of a serine esterase from rabbit liver in which DFP was found to bind not only to the active site serine but also to the histidine in the catalytic triad (Korza and Ozols, 1988). The serine proteases arc orders of magnitude less sensitive to OPs than are the serine esterases. 

Enzyme inhibition sensors are the most commonly reported enzyme-based biosensors for the detection of toxic compounds and heavy metal ions. The sensors are based on the selective inhibition of specific enzymes by classes of compounds or by the more general inhibition of enzyme activity. Most of the research carried out has been directed toward the detection of organophosphorus and carbamate insecticides and the triazine herbicides and metal ions analysis [72,73]. Several enzymes have been used in inhibition sensors for pesticides and heavy metal analysis using water, soil, and food samples including choline esterase, horseradish peroxidase, polyphenol oxidase, urease, and aldehyde dehydrogenase. 

Ethyl acetate is produced by the yeast, Saccharomyces cerevisiae, using the acetyl alcohol transferase enzyme. It links the acetate from the acetyl-coA with an ethanol molecule. The esters produced can be affected by esterases enzymes, because they hydrolyze the aliphatic and aromatic esters. These enzymes can hydrolyze lipids to obtain medium chain fatty acids (Moreno. 2009). Production of esters is related to the quantity of amino acids. When the nitrogen from the amino acids is high, fatty acids decrease and acyl transferase is not inhibited (Arrizon. 2001). 

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