Acetylcholinesterase, inhibition with phosphorylated

The OPPs inhibit acetylcholinesterase (AChE) by phosphorylating the esteratic site of the enzyme. As a result of AChE inhibition, ACh accumulates and binds to muscarinic and nicotinic receptors throughout the nervous system. Transformation of OPPs in the organisms takes place by conversion of the phosphorothioate (P=S) group to oxon (P=0) analogs. These oxo compounds are of concern because they are the activated forms of the OPPs, with a considerably stronger inhibition of acetylcholinesterase activity (27). 

Tphe search for insecticides with modes of action different from the A well-known acetylcholinesterase inhibition led us to uncouplers of oxidative phosphorylation (1, 2). An inherent advantage of such pesticides would be the absence of cross-resistance with organophosphorus compounds and chlorinated hydrocarbons. The number of commercial pesticides which are likely to act by uncoupling of oxidative phosphorylation is small. All of them can be regarded as derivatives of the... 

Recovery of phosphinylated acetylcholinesterase was also greatly enhanced in our studies of methyl parathion-resistant tobacco budworm larvae (Figure 5). Phosphinylated acetylcholinesterase recovery can be measured without the complication of "aging" which occurs with phosphorylated enzyme (Figure 6). Activity was totally inhibited with 4-nitrophenyl methyl(phenyl)phosphinate. Excess inhibitor was removed by solid phase extraction and recovering activity at 30°C was monitored in aliquots. We have confirmed this insensitivity with individual heads of adults from these strains and we are investigating inheritance of this trait. 

This process of aging is believed to be critical in the development of delayed neuropathy, after NTE has been phosphorylated by an OP (see Chapter 10, Section 10.2.4). It is believed that most, if not all, of the B-esterases are sensitive to inhibition by OPs because they, too, have reactive serine at their active sites. It is important to emphasize that the interaction shown in Fignre 2.11 occurs with OPs that contain an oxon group. Phosphorothionates, which contain instead a thion group, do not readily interact in this way. Many OP insecticides are phosphorothionates, but these need to be converted to phosphate (oxon) forms by oxidative desulfuration before inhibition of acetylcholinesterase can proceed to any significant extent (see Section 2.3.2.2). 

Obidoxime is an antidote used to treat poisoning with insecticides of the organophosphate type (p. 102). Phosphorylation of acetylcholinesterase causes an irreversible inhibition of ace-Ltillmann, Color Atlas of Pharmacology 2000 Thieme All rights reserved. Usage subject to terms and conditions of license. 

The first suggestion of a practical form of antidotal therapy came in 1949 from Hestrin, who found that acetylcholinesterase (AChE) catalyzed the formation of acetohydroxamlc acid when incubated with sodium acetate and hydroxylamine. Critical in vitro studies in the next decade led to the development of a practical approach to therapy. The crucial concept in these studies was the recognition that the compound formed when AChE reacted with a phosphorus ester was a covalent phosphoryl-enzyme Intermediate similar to that formed in the hydrolysis of acetylcholine. 3 Wilson and colleagues, beginning in 1951, demonstrated that AChE inhibited by alkyl phosphate esters (tetraethyl pyrophosphate, TEPP) could be reactivated by water, but that free enzyme formed much more rapidly in the presence of hydroxylamine. 0 21 Similar results... 

Diazinon toxicity results predominantly from the inhibition of acetylcholinesterase in the central and peripheral nervous system. The enzyme is responsible for terminating the action of the neurotransmitter, acetylcholine, in the synapse of the pre- and post-synaptic nerve endings or in the neuromuscular junction. However, the action of acetylcholine does not persist long as it is hydrolyzed by the enzyme, acetylcholinesterase, and rapidly removed. As an anticholinesterase organophosphate, diazinon inhibits acetylcholinesterase by reacting with the active site to form a stable phosphorylated complex which is incapable of destroying acetylcholine at the synaptic gutter between the pre- and post-synaptic nerve... 

As outlined in Figure 3, the hydrolysis of paraoxon by human serum A-esterase(s) is very similar to the phosphorylation of B-esterases, such as acetylcholinesterase, by paraoxon. Both reactions involve an initial binding of paraoxon to the enzyme, followed by a rapid conformational change that produces diethyl phosphate and p-nitrophenol from paraoxon. p-Nitrophenol is quickly released from the enzyme, leaving diethyl phosphate covalently bound to enzyme. At this point, A-esterase quickly releases diethyl phosphate as a result of interacting with a water molecule. However, B-esterases, such as acetylcholinesterase, retain the diethyl phosphate for a much longer period of time, thereby resulting in inhibition of the enzyme. 

These organic phosphates inhibit acetylcholinesterase by reacting with the active-site serine residue to form a stable phosphorylated derivative. They cause respiratory paralysis by blocking synaptic transmission at cholinergic synapses. 

The toxicity of the G and V agents is apparently due to their affect upon the transmission of nerve impulses in the body, due to their ability to react with and inhibit acetylcholinesterase (ChE) enzyme. For example, after a nerve impulse is transmitted across a nerve and muscle junction by acetylcholine, the latter is then rapidly hydrolyzed by ChE. However, in the presence of GB, for example, the hydrolytic sites on the enzyme are irreversibly blocked by phosphorylation, and these sites are no longer available for hydrolysis of the acetylcholine. The resulting buildup of acetylcholine acts to overstimulate the muscle, which eventually goes into spasm. Thus, the typical symptoms of poisoning by a nerve agent are due to its cholinergic activity. 

Other types of inhibitors may not be so tolerable. Organophosphorus compounds, used in nerve gases and weed killers (e.g., parathion), form a covalent irreversible bond with the active serine and permanently inactivate acetylcholinesterase. This is a type of suicide inhibition because the inhibitor reacts with the enzyme much like a substrate, but becomes blocked in the intermediate state where the enzyme-phosphoryl bond is stable, in contrast to the hydrolyzable enzyme-acetyl bond. These compounds are life-threatening. 

Butyrylcholinesterase occurs in the liver and at the motor endplates in muscle fibers and at synapses together with aeetylcholinesterase (Silver 1974). It is estimated that approximately 15% of total cholinesterase activity in the nervous system is due to the nonspecific cholinesterase activity in some of the white matter (Ecobichon and Joy 1982), and the synthesis of this nonspecific cholinesterase occurs in the liver. Several organophosphorus compounds can react and phosphorylate acetylcholinesterase carbamate ester compounds can carbamylate the enzyme, so both can inhibit the acetylcholinesterase. 

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