Question

1) Briefly explain how V-series chemical warfare agents work.

2) Briefly explain the role of glutamic acid E327, histidine H440 and serine S220 in acetylcholinesterase catalysis.

Answer 1

1.

            V-Series nerve agents are organophosphate esters that are used as chemical weapons. They are extremely potent acetylcholinesterase inhibitors. Biological effects include seizures, salivation, lacrimation, urination, diaphoresis, diarrhea, vomiting, miosis, and muscles spasms. As little as a few milligrams of some V-series agents can be lethal to humans.

            The V-series nerve agents were first discovered in 1952 by scientists in the United Kingdom researching organophosphate esters as pesticides. The five most well-known V-series nerve agents are VX, VE, VG, VM, and VR. V-series nerve agents are highly viscous and have low volatility; thus, they can persist in the environment and are difficult to wash away. They are oily liquids at room temperature. Some V-series agents can be deployed as binary agents, in which two non-toxic chemicals react together inside the weapon before deployment to form the chemical weapon. For example, isopropyl aminomethyl ethyl phosphorite and elemental sulfur react to form VX. V-series agents can be deployed as liquids or aerosols.

            V-series nerve agents inhibit acetylcholinesterase when the enzyme binds a phosphoryl group in the esteratic subsite. The role of the acetylcholinesterase enzyme is to break down acetylcholine in the synapse into acetate and choline, and the enzyme can degrade approximately 25,000 molecules of acetylcholine per second. When the acetylcholinesterase enzyme is inhibited, it causes acetylcholine to accumulate in the synapse and thus continue to stimulate the acetylcholine receptors. Salivation, lacrimation, urination, diarrhea, and vomiting are caused by the stimulation of muscarinic acetylcholine receptors in the parasympathetic nervous systems. Skeletal muscle symptoms are caused by the stimulation of nicotinic acetylcholine receptors. Nerve agents have also demonstrated the ability to inhibit other enzymes, including neurotoxic esterase (NTE).

            The only use of V-series nerve agents (other than for research) is a chemical weapon. Thus, patients exposed to these agents may present in large numbers and report a history consistent with a terrorist attack. Symptoms are identical to those of the organophosphate toxidrome and include seizures, salivation, rhinorrhea, lacrimation, urination, diaphoresis, diarrhea, vomiting, miosis, and muscles spasms. Other symptoms include bronchospasm, central apnea, and bradycardia. Most deaths occur because of respiratory failure from the combination of respiratory symptoms. Those who survive nerve agent exposure may experience insomnia, depression, anxiety, irritability, and impaired memory and judgment. Ocular symptoms such as miosis, dim vision, blurry vision, and eye pain may persist for several weeks following exposure. Long-term neurologic effects of nerve agents have been reported.

2.

            Acetylcholinesterase (AChE) is a hydrolase that hydrolyzes choline esters. It has a very high catalytic activity—each molecule of AChE degrades about 25,000 molecules of acetylcholine (ACh) per second, approaching the limit allowed by diffusion of the substrate. The active site of AChE comprises 2 subsites—the anionic site and the esteratic subsite.

            The esteratic subsite, where acetylcholine is hydrolyzed to acetate and choline, contains the catalytic triad of three amino acids: serine 200, histidine 440 and glutamate 327. The triad is of opposite chirality to that of other proteases.

The hydrolysis reaction of the carboxyl ester leads to the formation of an acyl-enzyme and free choline:

            These residues are arranged in such a way that the histidine and glutamate donate electrons to the serine, making it reactive. The serine is able to form a covalent bond with acetylcholine, reducing its carbonoxygen double bond. This species is termed the tetrahedral intermediate, in which the oxygen is charged. This ‘oxyanion’ is stabilised by interactions with backbone amide groups in an area termed the ‘oxyanion hole’. From this intermediate, the acetylcholine bond is broken, releasing choline and leaving acetylserine.

            Then, the acyl-enzyme undergoes nucleophilic attack by a water molecule, assisted by the histidine 440 group, liberating acetic acid and regenerating the free enzyme.

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