MECHANISM OF ACTION OF ACETYLCHOLINESTERASE IN THE BODY AND

CNS AND ITS RELATION TO THE CURRY CRISIS

Acetylcholine is an important neurotransmitter used in the
human body and is responsible for many processes
necessary to sustain life. The mechanism by which
acetylcholine works is called cholinergic neurotransmission,
and is depicted schematically in Figure 1. However, as is the
case with almost all drugs, overstimulation can occur leading
to complications such as bradycardia, muscle weakness and
seizures, among many others including death (Mangas et al.,
2016). To prohibit overstimulation, the body has evolved an
enzyme called acetylcholinesterase (AChE) a serine
hydrolase responsible for the metabolism of acetylcholine
and subsequently the termination of impulse cholinergic
neurotransmission. The enzyme is found mainly at Figure 1: Diagram of cholinergic transmission at a
chemical synapse. Calcium ions trigger the release
neuromuscular junctions (NMJ) and cholinergic brain synapses of acetylcholine (ACh) into the synaptic cleft,
opening ion channels a nd triggering a physiological
(olovi et al., 2013). AChE is one of the most efficient response in the postsynaptic neuron. AChE
metabolises ACh so that ion c hannels may close to
enzymes in nature with each molecule able to metabolise avoid overstimulation, after which case the
metabolite choline re-enters the presynaptic
25000 molecules of ACh per second (olovi et al., 2013), synapse via transport proteins to be reused to
make ACh a t a later time when required. A similar
however it still has its limitations unavoidable due to the very mechanism occurs at neuromuscular junctions,
where the difference is the response in the
laws of physics that dictate the mechanisms of organic postsynaptic neuron, which is muscle contraction
(Field and Wymore, 2014). (Taken from Field and
chemistry by which the enzyme is allowed to work in the first Wymore, 2014).

place. In order to understand these limitations, it must first be outlined how the enzyme
works to metabolise acetylcholine.

The structure of the AChE enzyme allows for cholinergic drugs including acetylcholine to bind
to a sequence of amino acids in the protein structure of the enzyme, as long as the drug is not
sterically or electronically hindered. The ligand binds to an anionic site, which has partial
negative charge and so binds to the electropositive quaternary amine of the choline moiety.
It also binds to an esteratic site where the sequence of amino acids resides and where the
significant chemical reactions takes place (ATSDR, 2007; olovi et al., 2013). A schematic

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 diagram of AChE is shown below in Figure 2.1, with relevant binding sites depicted in Figure
2.2.

Figure 2.1: D iagram of 3 dimensional structure o f AChE determined by Figure 2.2: Schematic of AChE binding sites (from olovi et a l., 2013)
X-ray crystallography (from Field a nd Wymore, 2014).

The active site of the enzyme is depicted in Figure 2.2, which resides 4 from the bottom of
the molecule. The anionic site is represented in the diagram collectively by the acyl pocket
and choline binding site. It is uncharged and lipophilic. The cationic substrate is not bound by
ionic means to an amino-acid in the anionic site, but by Van der Waal interactions with 14
aromatic amino-acid functional groups that line the site. Key amino-acids in this site are the
aromatic phenyl group-containing amino-acids phenylalanine and tryptophan, with
tryptophan 84 being critical to the activity of the enzyme (olovi et al., 2013). The esteratic
site comprises of the catalytic triad that is common among serine hydrolases the amino-
acids serine 200, histidine 440 and glutamate 327 (olovi et al., 2013).

The hydrolysis reaction of acetylcholine by AChE has been studied extensively experimentally,
and with the aid of quantum mechanics/molecular mechanics (QM/MM) simulations of
chemical reactions of the enzyme with acetylcholine and other ligands, mechanistic
information of AChE has been obtained (Zhou et al., 2010; Field and Wymore, 2014). It has
been discovered that human AChE metabolises acetylcholine in a two-step process: acylation,
followed by deacylation (Field and Wymore, 2014). The chemical reaction is shown
diagrammatically in Figure 3.

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Figure 3: C hemical mechanism by which acetylcholinesterase metabolises acetylcholine (from olovi et al., 2013).

In the acylation step, hydrolysis of the carboxyl ester of acetylcholine leads to the formation
of an acylated enzyme and a free choline metabolite (in the diagram above this is depicted by
the reaction in the first two steps). This is followed immediately by the nucleophilic attack of
a water molecule on the carbonyl carbon of the acyl-enzyme product of the previous step,
which is assisted by the histidine 440 group. This removes the acetate from the enzyme
complex, resulting in liberation of acetic acid and reactivation of AChE (olovi et al., 2013).

With the knowledge of the structure of this enzyme and how it works to metabolise
cholinergic ligands, it can be understood how inhibitors of AChE work. In the scenario of the
Curry Crisis, the children experienced symptoms of stomach ache, watery eyes, sweating,
vomiting, diarrhoea, muscle twitching, convulsions, loss of consciousness and death. These

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symptoms fit closely to those of acute organophosphate (OP) poisoning, which elicits its
effects via acetylcholinesterase inhibition. These symptoms are listed in Table 1 below.
Receptor Symptoms
Diarrhoea, urinary incontinence, miosis, bradycardia,
bronchoconstriction, bronchorrhoea, hypotension, increased
Muscarinic receptors (mAChR)
gastrointestinal motility, abdominal cramps, hypersalivation,
lacrimation

Hypertension, tachycardia, fibrillation, fasciculation, striated

Nicotinic receptors (nAChR)
muscle necrosis

Tremor, loss of movement coordination, seizures, central

Receptors in the CNS
depression of respiration, coma, death

Table 1: Acute symptoms of OP poisoning a t their respective receptor a s a result of AChE inhibition a nd subsequent
overstimulation of postsynaptic c holinergic receptors (taken from Mangas e t al., 2016).

The children reported the difference in taste of the new curry, and this was most likely due
to the change in cooking oil used, suggesting cross-contamination of the new ingredient with
foreign substances including insecticides could be the cause of the symptoms. The
poisoning could have also perhaps been caused by the contamination of cooking utensils such
as the pots and containers used to dish out food, if these were exposed to insecticides prior
to the cooking and distribution of the curry. For this reason, it shall be assumed that the
children in this scenario suffered from insecticide poisoning.

Insecticides come in mainly two forms: carbamates and organophosphates, which are popular
mainly due to their effectiveness and relatively low price (Mangas et al., 2016). The general
chemical structures of carbamates and organophosphates are depicted respectively in Figure
4.1 and Figure 4.2. Both figures are taken from olovi et al., 2013.

Figure 4.1: General structural formula for carbamates Figure 4.2: General structural formula for organophosphates

The letters for both structural formulae above denote different atoms that may make up the
molecule. X may be oxygen or sulfur (in which case the carbamate is a thiocarbamate) and R1,
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R2 and R3 are different structural groups which may be hydrogen, metal atoms, or (usually)
organic substituents. In a carbamate, insecticide activity is induced if R1 is a methyl group and
R2 is a hydrogen (olovi et al., 2013). Since both classes of molecule inhibit AChE, they both
elicit similar toxic acute responses with the difference being that carbamate insecticides do
not form permanent covalent bonds with the enzyme, as OP insecticides do. Carbamate
molecules are able to spontaneously hydrolyse from the enzyme-carbamate complex in much
the same way that acetate can, however the process takes a much longer time (approximately
30 40 minutes, whereas with acetylcholine deacylation is immediate) (olovi et al., 2013).
In the Curry Crisis, however, the childrens symptoms lasted longer than 30 40 minutes
and were more severe, indicating organophosphate poisoning was the cause.

The mechanism by which AChE metabolises OPs is as such. In much the same way as acylation,
the enzyme reacts with the drug in a substitution SN2 reaction, but in this case it becomes
phosphorylated. This is because the structure of OPs closely resemble the structure of
acetylcholine (olovi et al., 2013). The next step differs in that deacylation occurs quickly,
whereas dephosphorylation occurs much more slowly (on the order of days) and that is if
the OP does not undergo aging, which increases their toxicity further. This is an enzyme-
mediated process, in which a covalent bond is formed irreversibly between the phosphorus
atom and the enzyme due to the removal of an alkyl side group of the OP caused by
nucleophilic attack of a hydroxyl anion, resulting in a negative charge on the oxygen atom
that was linked to the alkyl group (Field and Wymore, 2014). This makes the enzyme much
more resistant to reactivation, as the formation of a salt bridge is promoted between the
protonated histidine and the negatively charged oxygen atom bound to the phosphorus atom.
Conformational and stability changes of the enzyme also occur during (and as a result of) this
reaction (Curtil and Masson, 1993). The result is irreversible and the enzyme is permanently
inactivated. In a way, the enzyme catalyses its own demise. This process is illustrated on the
following page in Figure 5 with the example of the OP soman, which is a nerve agent but acts
in a similar way to insecticide agents.

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Figure 5: Process by which the OP soman inhibits and ages AChE. It works in the same way as insecticide OPs, but the aging mechanism
occurs much more rapidly. In the case for soman, AChE causes dealkylation to occur, eliminating the alkyl moiety leaving the enzyme
irreversibly phosphorylated and the alkyl side chain as a carbenium cation, ready to react with other molecules in solution (Source: Field
and Wymore, 2014).

The irreversible formation of the phosphorylated enzyme
causes AChE inactivation. As a result, acetylcholine levels
build up in the synaptic cleft as it can no longer metabolise
the neurotransmitter, causing overstimulation of
cholinergic receptors and leading to the symptoms listed
in Table 1 and the ones experienced by the children in the
Curry Crisis (olovi et al., 2013). Depicted in Figure 6.1
and Figure 6.2 are chemical structures of various OPs. The
chemicals in Figure 6.1 are all insecticides and in Figure 6.2
paraoxon and diisopropyl fluorophosphates (DFP) are OPs
used in medicine and as insecticides and sarin, soman, VX
and tabun are all examples of lethal nerve agents.

Figure 6.1 (above): Chemical structures of various OP
insecticides. (Taken from olovi et a l., 2013).

Figure 6.2 (left): Chemical structures of various OPs. The
structures shown in red are the leaving groups once the
drug undergoes hydrolysis (Field and Wymore, 2014).
(Taken from Field a nd Wymore, 2014)

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In conclusion, acetylcholinesterase is a very important enzyme in the body, as it is responsible
for the metabolism of acetylcholine in synaptic clefts so that overstimulation of receptors
does not occur. Through analysis of the Curry Crisis scenario, the symptoms of the children
affected closely matched the symptoms induced by overstimulation of cholinergic receptors
as a result of acetylcholinesterase inhibition. It was hypothesised that insecticide poisoning
was the cause, with two main culprits identified due to their availability in the context of the
Curry Crisis: carbamates and organophosphates. The childrens symptoms lasted longer and
were more severe than would be expected of a carbamate pesticide, leaving
organophosphate poisoning as the remaining logical cause. Investigation of the pharmacology
and organic chemistry involved in the metabolism of acetylcholine and organophosphates
revealed that AChE is easily phosphorylated by OPs as they share a similar structure to
endogenous cholinergic neurotransmitters though it is much more difficult for the
phosphorylated enzyme to then be hydrolysed in order to reactivate it. Due to these reasons,
the conclusion was made that the children were most likely experiencing organophosphate
poisoning.

References
(2007). Cholinesterase Inhibitors - Management of the Cholinergic Toxidrome - Medications:
2-PAM (2-Pyridine Aldoxime Methylchloride) (Pralidoxime) | ATSDR - Environmental
Medicine & Environmental Health Education - CSEM. Available at:
https://www.atsdr.cdc.gov/csem/csem.asp?csem=11&po=23

Colovic, M., Krstic, D., Lazarevic-Pasti, T., Bondzic, A., and Vasic, V. (2013).
Acetylcholinesterase Inhibitors: Pharmacology and Toxicology. Current Neuropharmacology
11: 315-335.

Curtil, C., and Masson, P. (1993). [Aging of cholinesterase after inhibition by

organophosphates]. Annales Pharmaceutiques Francaises 51: 63-77.

Field, M., and Wymore, T. (2014). Multiscale modeling of nerve agent hydrolysis
mechanisms: a tale of two Nobel Prizes. Physica Scripta 89: 108004.

Mangas, I., Vilanova, E., Estvez, J., and Frana, T. (2017). Neurotoxic Effects Associated
with Current Uses of Organophosphorus Compounds.

Zhou, Y., Wang, S., and Zhang, Y. (2010). Catalytic Reaction Mechanism of
Acetylcholinesterase Determined by BornOppenheimer Ab Initio QM/MM Molecular
Dynamics Simulations. The Journal Of Physical Chemistry B 114: 8817-8825.

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