Which enzyme destroys acetylcholine

As a potent AChE inhibitor, this therapeutic agent reduces ACh hydrolysis rate, and thereby increases its level in damaged neurosynaptic clefts improving nerve impulse transmission. Besides, pyridostigmine Fig. Consequently, it is applied as a prophylactic against nerve agent intoxication [ 77 - 79 ]. Furthermore, rivastigmine Fig. OPs are esters or thiols derived from phosphoric, phosphonic, phosphinic or phosphoramidic acid Fig.

R 1 and R 2 are aryl or alkyl groups that are bonded to the phosphorus atom either directly forming phosphinates , or through an oxygen or sulphur atom forming phosphates or phosphothioates. In some cases, R 1 is directly bonded to the phosphorus atom, and R 2 is bonded to an oxygen or sulphur atom forming phosphonates or thiophosphonates.

In phosphoramidates, at least one of these groups is —NH 2 un-, mono- or bi-substituted , and the atom double-bonded with phosphorus is either oxygen or sulphur. The —X group, also binding to the phosphorus atom through oxygen or sulphur atom, may belong to a wide range of halogen, aliphatic, aromatic or heterocyclic groups.

The OPs exert their main toxicological effects through non-reversible phosphorylation of esterases in the central nervous system [ 81 , 82 ]. The acute toxic effects are related to irreversible inactivation of AChE [ 82 ]. Actually, OPs are substrate analogues to ACh, and like natural substrate enter the active site covalently binding to serine —OH group. As in acetylation, OP is split and the enzyme is phosphorylated Fig.

While the acyl enzyme is quickly hydrolyzed to regenerate the free enzyme, dephosphorylation is very slow on the order of days , and phosphorylated enzyme cannot hydrolyze the neurotransmitter [ 83 ]. The inhibition of the enzyme leads to accumulation of ACh in the synaptic cleft resulting in over-stimulation of nicotinic and muscarinic ACh receptors and impeded neurotransmission. The typical symptoms of acute poisoning are agitation, muscle weakness, muscle fasciculations, miosis, hypersalivation, sweating.

Irreversible inhibition occurs in two steps; the first one is fast, short term reversible enzyme inactivation, and its influence is dominant in the begining of the inhibition. The next step is slow irreversible inhibition producing a very stable enzyme-inhibitor complex phosphorylated enzyme -inhibitor is covalently bonded to the enzyme [ 88 ]. Time dependent irreversible inhibition can be described by the equation:. Progressive development of inhibition produced by reaction of AChE with different concentrations of diazoxon plotted as semi logarithmic curve in accordance with Equation 1.

Reproduced from [ 90 ]. The dependence of kapp upon the concentration of diazoxon 1 , chlorpyrifos-oxon 2 and chlorpyrifos 3 , inset plotted as reciprocals in accordance with Equation 2. Effective OPs have the following structural features: a terminal oxygen connected to phosphorus by a double bond oxo form , two lipophilic groups —R 1 , —R 2 bonded to the phosphorus, and a good leaving group —X bonded to the phosphorus Fig.

OPs can produce delayed neurotoxic effect in humans and chickens, called OP induced delayed neuropathy. It is associated with phosphorylation and further dealkylation aging Fig.

The symptoms of this neuropathy are paralysis and ataxia, and appear between 14 and 24 days after the poisoning [ 67 , 70 , 71 ]. The majority of OPs have been commonly used as nonspecific insecticides for over fifty years, to control a variety of insects in agriculture and the household environment.

The synthesis of OP pesticides in large quantities started after World War II, and parathion was among the first marketed, followed by malathion and azinphosmethyl. Commonly used OP insecticides have included ethyl parathion, malathion, methyl parathion, chlorpyrifos, diazinon, dichlorvos, phosmet, fenitrothion, tetrachlorvinphos, azinphos methyl, pirimiphos-methyl, dimethoate, phosalone Fig.

In the s organochlorine insecticides DDT, dieldrin, aheptachlor were banned because of their persistence and accumulation in the environment, and replaced by more degradable OPs. Actually, OP insecticides in the environment undergo the natural degradation pathway including mainly homogeneous and heterogeneous hydrolysis especially at high pH enhanced by the presence of dissolved metals, humic substances, microorganisms and other compounds present in soil [ 94 - 96 ].

OP degradation processes also occur in chemical treatments for purification of polluted waters, generally referred as advanced oxidation processes, as well as throughout the enzymatic reactions in birds, fish, insects and mammals. Degradation studies revealed different kinetics, mechanisms and transformation products, suggesting complete mineralization of the starting compound usually thio form , but forming toxic break down products as well [ 89 , 97 - ].

Actually, oxidation and isomerisation reaction products were reported as much more potent AChE inhibitors compared to the starting thio OPs, while hydrolysis products do not noticeably affect the enzyme activity. Although OPs insecticides degrade rapidly, that made them an attractive alternative to the organochloride pesticides, they have greater acute toxicity, posing risks to people who may be exposed to large amounts - workers employed in the manufacture and application of these pesticides.

OPs are one of the most common causes of poisoning worldwide occurring as a result of agricultural use, suicide or accidental exposure. OP pesticides can be absorbed by all routes, including inhalation, ingestion, and dermal absorption [ ]. Their toxicity is not limited to the acute phase, but chronic effects have long been noted. Actually, repeated or prolonged exposure to OPs may result in the same effects as acute exposure including the delayed symptoms.

The effects, reported in workers repeatedly exposed, include impaired memory and concentration, disorientation, severe depressions, irritability, confusion, headache, speech difficulties, delayed reaction times, nightmares, sleepwalking and drowsiness or insomnia. Influenza-like condition with headache, nausea, weakness, loss of appetite, and malaise has also been reported [ ]. Neurotransmitters such as ACh are profoundly important in the brain's development, and many OPs have neurotoxic effects on developing organisms, even from low levels of exposure, causing various diseases of nervous and immune system [ 85 , ].

Oxo forms of OP insecticides,are highly, approximately equally toxic to warm-blooded as well as cold-blooded organisms. On the other hand, thio forms are converted into the oxo forms by mixed function oxidases. The activation proceeds in cold-blooded organisms but this is not common in warm-blooded organisms where dealkylation into non toxic compounds takes place [ 51 , ].

Thus, numerous derivatives of highly toxic insecticides have been prepared to reduce the toxicity towards warm-blooded organisms and retain toxicity to insects, thereby enhancing their specificity. The examples of effective, commonly used OP insecticides, and relative safe for warm-blooded organisms are: malathion, chlorpyrifos, fenitrothion, pirimiphos-methyl, dimethoate, phosalone [ 51 ].

Nowadays, the common use of OP insecticides results in their accumulation, environmental pollution and acute and chronic poisoning events [ ]. For this reason, the use of OP insecticides has to be strictly controlled and restricted.

Accordingly, the majority of countries have strong regulations on the application of pesticides; e. Also, the applied insecticides and their by-products in the environment, water and food are monitored applying different bioanalytical techniques [ ]. Nerve agents of OP group include tabun, sarin, soman, cyclosarin and VX. Sarin, soman and cyclosarin are phosphonofluoridates, and VX is a phosphonothioate Fig.

Soman has four, while sarin and VX have two isoforms, which significantly differ in toxicity and irreversible AChE inactivation rate. Based on the acute toxicity, VX is the most toxic compound among all the nerve agents [ 67 ]. The developing and production of these extremely toxic nerve agents started in the s, and later used in wars and by terrorists on several occasions.

As chemical weapons, they are classified as weapons of mass destruction by the United Nations, and their production and stockpiling was outlawed by the Chemical Weapons Convention. Acute poisoning by a nerve agent leads to contraction of pupils, profuse salivation, convulsions, involuntary urination and defecation, and eventual death by asphyxiation as control is lost over respiratory muscles.

Some nerve agents are readily vaporized or aerosolized and the primary portal of entry into the body is the respiratory system. Nerve agents can also be absorbed through the skin, requiring that those exposed to such agents wear a full body suit in addition to a respirator [ ].

Moreover, the effects of nerve agents are very long lasting and cumulative increased by successive exposures , and survivors of nerve agent poisoning usually suffer chronic neurological damage that can lead to continuing psychiatric effects [ ].

OPs, except their use as toxic compounds, have been applied in ophthalmology as therapeutic agents in the treatment of chronic glaucoma, an eye disease in which the optic nerve is damaged in a characteristic pattern. The disease is associated with increased fluid pressure in the eye, and can permanently damage vision in the affected eye s and lead to blindness if left untreated [ ].

These medical useful OPs include diisopropyl fluorophosphate and echothiophate. It is known as fluostigmine and dyflos in such uses. It exerts ocular side effects mainly associated with its AChE inhibitory properties, and ability to induce delayed peripheral neuropathy [ 67 , ]. Echothiophate phospholine Fig. It is used as an ocular antihypertensive in the treatment of chronic glaucoma and, in some cases, accommodative esotropia.

Its application is local eye drops , and the effects can last a week or more. The drug is available under several trade names such as phospholine iodide. Adverse effects include muscle spasm and other systemic effects [ ].

OP compounds may be used in the therapy of neurological damages such as AD and Parkinson's disease. The example is trichlorfon metrifonate Fig. The primary target of OP action is AChE, and the main mechanism of toxicity in acute OP exposure involves the specific irreversible inhibition of this enzyme activity in the nervous system and blood, manifesting as a cholinergic crisis with excessive glandular secretions and weakness, miosis and fasciculation of muscle, which may lead to death [ , , ].

Additionally, many studies suggest that both acute and chronic intoxication disturb the redox processes changing the activities of antioxidative enzymes and causing enhancement of lipid peroxidation in many organs, and there is little correlation between organ damage and the degree of OP induced AChE inhibition [ - ]. Indeed, in acute, and rather subchronic or chronic OP exposition, induction of oxidative stress has been reported as the main mechanism of its toxicity [ ].

Oxidative stress is defined as an imbalance between the production of free radicals — reactive oxygen species ROS and the antioxidant defense system — enzymatic and non-enzymatic. The ROS may be generated as the result of the metabolism of OPs by cytochrome Ps, monooxygenases that catalyze oxidation by addition of one atom of molecular oxygen into the substrate OP by electron transport pathway [ ]. There is some evidence that OPs may affect liver, kidney, muscles, immune, and hematological system, causing many human body disorders [ - ].

Also, some findings indicate oxidative stress as an important pathomechanism of neurological disorders such as AD and Parkinson's disease, as well as of cardiovascular diseases [ , , ]. The highly reactive free radicals attack DNA resulting in single and double strand breaks, as well as oxidative damage to sugar and base residues that can later be converted to strand breaks [ ]. On the other hand, phosphorus moiety in the OPs appears to be a good substrate for nucleophilic attack leading to phosphorylation of DNA which is an instance of DNA damage [ ].

Some reported studies indicate the increase in chromosomal aberrations CA , micronuclei MN and sister chromatid exchanges SCE , as the markers of cytogenetic damage, in cultured lymphocytes isolated from peripheral blood taken from exposed individuals. Thus, cytogenetic damage in circulating lymphocytes has been widely used as a biomarker of exposure and effects of pesticides [ , ].

It has been reported that AChE non-inhibiting OP decomposition products exert stronger genotoxic potency compared to the parent compound [ 99 ], suggesting that the risk of genotoxicity from some insecticides might be appreciably greater than that predicted from standard toxicity tests [ ].

Moreover, DNA damage leads to genomic instability that may result in mutagenesis and carcinogenesis [ ]. Some epidemiological studies demonstrate cancer risk due to pesticides exposure [ - ], while The United States Environmental Protection Agency lists parathion as a possible human carcinogen [ ].

The mechanism of OP insecticides action is based on the irreversible inhibition of AChE in an insect body, resulting in the disrupted neuronal transmission and the consequent death.

However, the OPs are not selective for insect species, but they have the same mechanism of action for the warm-blooded organisms including humans that may be also intoxicated. Actually, OPs irreversibly inhibit human AChE in Ser leading to the cholinergic crisis which manifests as the muscarinic lacrimation, salivation, miosis , nicotinic neuromuscular blockade or central breath depression symptoms, and the organism death in the case of untreated OP intoxication [ 52 ].

OP poisoning can be treated non-pharmacologically and pharmacologically [ ]. The non-pharmacologic treatment includes resuscitation, oxygen supply or decontamination depending on the OP entrance to the human body e. Parasympatolytics usually atropine, Fig. The causal treatment comprises AChE reactivators that, unlike the symptomatic drugs, regenerate the enzyme native function by cleaving OP moiety from AChE serine active site. The mechanism of AChE reactivation is based on the nucleophilic attack of the reactivator hydroxyiminomethyl oxime moiety towards the OP moiety of the phosphorylated AChE.

However, the inactivated phosphorylated enzyme can be dealkylated Fig. Therefore, the oxime reactivators should be administered rapidly after the OP poisoning [ ]. The commercially available oxime reactivators - pralidoxime, methoxime, trimedoxime, obidoxime, asoxime Fig. Additionally, some of them exerted good effects in the restoration of inhibited cholinesterases by OP insecticides as well [ ]. Although pralidoxime was the first synthesized and most common used causal drug, its capability to regenerate the inhibited enzyme activity is not satisfactory.

On the contrary, bisquaternary oximes, trimedoxime and obidoxime exhibited very good abilities in the treatment of OP insecticides poisoning, whereas asoxime was found to be effective for nerve agent induced intoxication and without the meaningful reactivation of the inhibited AChE by OP insecticides.

Moreover, obidoxime, combined with atropine and diazepam, has demonstrated positive results in the clinical trials [ , ].

Furthermore, among synthesized and evaluated oxime reactivators, some novel compounds e. K, BTM, TMB-4, BT possess, related to the commercially available reactivators, both better reactivation capabilities against different OP insecticides and significantly decreased toxicity tested using in vitro and in vivo animal models [ - ]. Carbamates and OPs are apolar compounds accumulating in fatty tissues, and can be eliminated by their conversion to water soluble compounds.

The most effective way to increase the water solubility of these toxic compounds is hydrolysis to much more water soluble metabolites that may be removed in urine. Although these insecticides are able to hydrolyze spontaneously especially at high pH, the main route of their detoxification is enzymatic degradation by hydrolases generating less toxic metabolites. Carbamates are most effectively decomposed by carboxylesterases CESs , the esterases capable to hydrolyze carboxyl esters [ ].

The mechanism of the CESs catalyzed hydrolysis of carboxyl esters Fig. Subsequently, the acylated intermediate is decomposed by nucleophilic water attack to the corresponding carboxylic acid and the free active CES participating in the new catalytic cycle. Highest CESs concentrations have been found in serum and liver of mammals [ ]. Since the carbamates are structurally similar to carboxyl esters, these insecticides are susceptive to CESs catalyzed hydrolysis.

The mechanism of carbamates induced inhibition of CESs is similar to the mechanism of hydrolysis of the enzyme natural substrates. The additional step is the final rapid degradation of carbamic acid to carbon dioxide and the corresponding amine Fig. Moreover, oxidized metabolites of the parent carbamate insecticides can also be decomposed by CESs due to the intact carbamic ester bond [ 71 ]. The metabolism of carbamates depends on their chemical structure and animal species.

So, the hydrolysis of carbaryl in rabbit serum is significantly more efficient than chicken and human serum activity. Also, CESs with the ability to hydrolyze carbamates have been found in some bacteria Blastobacter, Arthrobacter, Pseudomonas, Achromobacter, Micrococcus [ 71 , ]. Additionally, some findings have demonstrated the capacity of serum albumin to decompose N -methylcarbamates and some OPs, and its hydrolyzing potency differs among species.

Generally, mammals exert higher hydrolyzing activity than birds [ 71 , , ]. OPs are detoxified through oxidation and hydrolysis. Actually, in mammals especially in liver and serum there is a pool of CESs of unknown physiological role, named B-esterases that both hydrolyze carboxyl esters and being inhibited by OPs.

Different from A-esterases being capable to hydrolyze carboxyl esters but not inhibited by OPs, B-esterases may be involved in the detoxification of OPs, and carbamates as well [ ]. While OP and carbamate insecticides exhibit major toxic effects through phosphorylation and carbamylation, respectively, of AChE and neuropathy target esterase inactivating these CESs, the inhibition of B-esterases is not associated with evident toxic effects. The mechanism of B-esterases inhibition by OPs is analogous to that induced by carbamates Fig.

The difference is in the final step; the phosphorylated enzyme cannot be reactivated by water, and cannot release the free active enzyme Fig. In this detoxification system that removes the insecticides from the media, each molecule of CESs scavenges at least one molecule of the toxic compound before this reaches targets in the nervous system [ 71 , ].

However, much more efficient route of OPs detoxification is hydrolysis by PTEs, esterases of an unknown physiological role. The resulting metabolites are less toxic and more polar than the parent OP, and therefore do not accumulate in fatty tissues and are eliminated in urine. PTEs have been found in various biological tissues of mammals, fish, birds, molluscs and bacteria.

Although these detoxification enzymes are located in a few tissues of mammals, higher PTEs activity levels have been detected in serum and liver [ , ]. In the central nervous system, ACh is found primarily in interneurons, shown in Figure A few important long-axon cholinergic pathways have also been identified. Noteworthy is the cholinergic projection from the nucleus basalis of Meynert in the basal forebrain to the forebrain neocortex and associated limbic structures, represented by the black pathway in Figure Degeneration of this pathway is one of the pathologies associated with Alzheimer's disease.

There is also a projection from the medial septal and diagonal band region to limbic structures blue. Most subcortical areas are innervated by neurons from the ponto-mesencephalic region purple in Figure Click on the region of the cell describing these processes to learn more about each one. As is the case for all nerve terminal proteins, CAT is produced in the cholinergic cell body and transported down the axon to the nerve endings.

Both CAT and ACh may be found throughout the neuron, but their highest concentration is in axon terminals. The rate-limiting steps in ACh synthesis are the availability of choline and acetyl-CoA.

During increased neuronal activity the availability of acetyl-CoA from the mitochondria is upregulated as is the uptake of choline into the nerve ending from the synaptic cleft. As will be described later, the inactivation of ACh is converted by metabolism to choline and acetic acid. Consequently much of the choline used for ACh synthesis comes from the recycling of choline from metabolized ACh. Another source is the breakdown of the phospholipid, phosphatidylcholine.

One of the strategies to increase ACh neurotransmission is the administration of choline in the diet. However, this has not been effective, probably because the administration of choline does not increase the availability of choline in the CNS.

The majority of the ACh in nerve endings is contained in clear as viewed in the electron microscope um vesicles. A small amount is also free in the cytosol. Vesicle-bound ACh is not accessible to degradation by acetylcholinesterase see below.

The uptake of ACh into storage vesicle occurs through an energy-dependent pump that acidifies the vesicle. No useful pharmacological agents are available to modify cholinergic function through interaction with the storage of ACh. Interestingly, the gene for VAChT is contained on the first intron of the choline acetyltransferase gene. This proximity implies the two important cholinergic proteins are probably regulated coordinately. You will recall that the miniature endplate potentials and the quantal release in response to action potentials at the neuromuscular junction are due to the release of packets of ACh from individual storage vesicles Chapter 5.

Many toxins are known that interfere with these processes and are effective in preventing ACh secretion. The examples in Figure There are two broad classes of cholinergic receptors: nicotinic and muscarinic. This classification is based on two chemical agents that mimic the effects of ACh at the receptor site nicotine and muscarine.

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Toggle navigation PDB Educational portal of. Molecule of the Month. Acetylcholinesterase Acetylcholinesterase stops the signal between a nerve cell and a muscle cell Download high quality TIFF image. Every time you move a muscle and every time you think a thought, your nerve cells are hard at work. They are processing information: receiving signals, deciding what to do with them, and dispatching new messages off to their neighbors.

Some nerve cells communicate directly with muscle cells, sending them the signal to contract. Other nerve cells are involved solely in the bureaucracy of information, spending their lives communicating only with other nerve cells.

But unlike our human bureaucracies, this processing of information must be fast in order to keep up with the ever-changing demands of life. Nerves communicate with one another and with muscle cells by using neurotransmitters. These are small molecules that are released from the nerve cell and rapidly diffuse to neighboring cells, stimulating a response once they arrive.

Many different neurotransmitters are used for different jobs: glutamate excites nerves into action; GABA inhibits the passing of information; dopamine and serotonin are involved in the subtle messages of thought and cognition.

The main job of the neurotransmitter acetylcholine is to carry the signal from nerve cells to muscle cells. When a motor nerve cell gets the proper signal from the nervous system, it releases acetylcholine into its synapses with muscle cells. There, acetylcholine opens receptors on the muscle cells, triggering the process of contraction. Of course, once the message is passed, the neurotransmitter must be destroyed, otherwise later signals would get mixed up in a jumble of obsolete neurotransmitter molecules.

Basically, a molecule is cleaved into two parts by reacting with water; part of the molecule binds to the H of water and the other part of the molecule binds to the OH of water, thus splitting the molecule and using up a molecule of water in the process.

When esters are hydrolyzed by water, the bond between the O atom and the C chain breaks. Since acetylcholine has an ester group, it is especially vulnerable to hydrolysis 6 by water Figure 8.

In this case, the ester bond which connects an acetyl group to the C chain, is hydrolyzed to give acetic acid vinegar and choline.

These 2 compounds recombine inside the nerve terminal to synthesize new acetylcholine. Acetylcholinesterase is the catalyst that makes this happen very quickly Figure 9. First, acetylcholine binds to the enzyme at 2 different sites; its acetyl group part of the ester binds to a specific OH group from the amino acid, serine in one place on the enzyme and the other end of acetylcholine binds to the enzyme in another place.

These bonds are ionic 7 or electrostatic in nature; opposite charges on the acetylcholine and the enzyme attract each other.