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Acetylcholinesterase and Butyrylcholinesterase: Substrate Specificity and Inhibition
Introduction
Cholinergic compounds are physiologically important due to their action on acetylcholine
receptors. This action must be regulated to avoid over-stimulation of, for instance, the
nervous system (Brown, 2006). Hence, metabolism of these chemicals through hydrolysis by
cholinesterases including acetylcholinesterase and butyrylcholinesterase is essential. An
understanding of the substrate selectivity of these cholinesterases and how their hydrolysing
activity is inhibited can be employed clinically to treat neurological disorders.
Therefore, the substrate specificity of acetylcholinesterase and butyrylcholinesterase was
examined first. These enzymes differ in selectivity for substrates based on structure and
conformational freedom (Trevor et al, 1978). Acetylcholine, butyrylcholine, and the
acetylcholine-derived choline esters methacholine, carbachol, benzoylcholine and
suxamethonium (Dale et al., 2007) were added to these enzymes. The hydrolysis of these
cholinergic substrates produces acetic acid. The subsequent increase in pH was modelled
experimentally with an indicator (Discipline of Pharmacology, 2010). Hence a colour change
demonstrated metabolism, with elevated rates of colour change denoting increased selectivity
of the enzyme for the substrate. The action of acetylcholinesterase on acetylcholine and
butyrylcholinesterase on butyrylcholine were set as 100% velocity.
Furthermore, preventing this metabolism is clinically beneficial in neurological diseases such
as Alzheimer’s. Anticholinesterases inhibit this cholinesterase metabolism through formation
of inactive complexes of varying stabilities (Burgen, 1949). Hence, the effects of the
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inhibitors edrophonium (short-acting), physostigmine and neostigmine (medium-acting), and
malathion (irreversibly acting) on the hydrolysis of acetylcholine and butyrylcholine were
observed (Dale et al., 2007). Atropine and carbachol action, a cholinergic antagonist and
agonist respectively, were also investigated.
The aims of this practical were to demonstrate the selectivity of acetylcholinesterase and
butyrylcholinesterase for various cholinergic compounds, and to examine the inhibitory
action of various anticholinesterases. It was hypothesised that an increase in the relative
velocity (RV) of colour change would be observed when substrates are in the presence of a
cholinesterase for which they are more structurally selective. Furthermore, the RV was
anticipated to decrease as the duration of action of the inhibitor specific for the cholinesterase
in use increased.
Results
0.0 0.0
100.0 100.0
2.6 1.1 0.0
195.0
0.00.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
160.0
180.0
200.0
1 2 3 4 5 6 7 8 9
Condition
Relative Velocity (%)
Figure 1: Relative percentage velocity of colour change of various cholinergic substrates and water in
acetylcholinesterase or water. In the presence of water and Tris buffer, in condition (1) 100 mM
acetylcholine was added to water and mixed, while in the remaining conditions various 100 mM
substrates or water were individually added to acetylcholinesterase – condition (2) water; (3)
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acetylcholine; (4) acetylcholine; (5) butyrylcholine; (6) benzoylcholine; (7) carbachol; (8) methacholine;
(9) suxamethonium. Both the degree and time of colour change were recorded, which were used to
calculate the relative percentage velocity of colour change of the each condition relative to the mean
velocity of colour change of conditions 3 and 4.
In Figure 1, methacholine produced the fastest relative velocity of colour change in
acetylcholinesterase (RV=195%), followed by acetylcholine (RV=100%), butyrylcholine
(RV=2.6%) and benzoylcholine (R=1.1%). Furthermore, carbachol, suxamethonium,
condition 1 (acetylcholine with water) and condition 2 (water with acetylcholinesterase) did
not produce any colour change.
0.0 0.0
100.0 100.0
33.8
104.2
21.9
1.3 0.00.0
20.0
40.0
60.0
80.0
100.0
120.0
1 2 3 4 5 6 7 8 9
Condition
Relative Velocity (%)
Figure 2: Relative percentage velocity of colour change of various cholinergic substrates and water in the
presence of butyrylcholinesterase and water. In the presence of water and Tris buffer, in condition (1) 100
mM Butyrylcholine was added to water and mixed, while in the remaining conditions various 100 mM
substrates or water were individually added to ButyrylcholineE – condition (2) water; (3) butyrylcholine; (4)
butyrylcholine; (5) acetylcholine; (6) benzoylcholine; (7) carbachol; (8) methacholine; (9) suxamethonium. Both
the degree and time of colour change were recorded, which were used to calculate the relative percentage
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velocity of colour change of the each condition relative to the mean velocity of colour change of conditions 3
and 4.
In Figure 2, benzoylcholine produced the fastest RV in butyrylcholinesterase (104.2%),
followed by butyrylcholine (100%), acetylcholine (33.8%), carbachol (21.9%) and
methacholine (1.3%). However, suxamethonium, condition 1 (butyrylcholine with water) and
condition 2 (water with butyrylcholinesterase) did not produce any colour change.
7.4 10.5
25.0
139.3
121.9
17.1
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
160.0
Physostigmine Neostigmine Edrophonium Malathion Carbachol(10mM)
Inhibitor
Relative Velocity (%)
Figure 3: Relative percentage velocity of colour change of acetylcholine in acetylcholinesterase in the
presence of various inhibitors. Various inhibitors were added to Tris buffer and Acetylcholinesterase, and 100
mM acetylcholine was then added to this mixture. Both the degree and time of colour change were recorded,
which were used to calculate the relative percentage velocity of colour change of the each condition relative to
the mean velocity of colour change of acetylcholine in the presence of acetylcholinesterase.
The RV for the hydrolysis of acetylcholine by acetylcholinesterase (100%) was increased to
139.3% in the presence of 50mM malathion, and to 121.9% in 5mM atropine (Figure 3). It
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was, however, decreased in 80mM edrophonium (25.0%), 10mM carbachol (17.1%), 5mM
neostigmine (10.5%), and 5mM physostigmine (7.4%)(Figure 3).
5.7 2.6
89.3
113.6
89.3
9.8
0.0
20.0
40.0
60.0
80.0
100.0
120.0
Physostigmine Neostigmine Edrophonium Carbachol(10mM)
Inhibitor
Relative Velocity (%)
Figure 4: Relative percentage velocity of colour change of butyrylcholine in butyrylcholinesterase in the
presence of various inhibitors. Various inhibitors were added to Tris buffer and butyrylcholinesterase, and 100
mM butyrylcholine was then added to this mixture. Both the degree and time of colour change were recorded,
which were used to calculate the relative percentage velocity of colour change of each condition relative to the
mean velocity of colour change of butyrylcholine in the presence of butyrylcholinesterase.
In Figure 4, the RV for the hydrolysis of butyrylcholine by butyrylcholinesterase (100%) was
increased in the presence of 50mM malathion (113.6%). It was, however, decreased in the
presence of 80mM edrophonium (89.3%), 5mM atropine (89.3%), 10mM carbachol (9.8%),
5mM physostigmine (5.7%), and 5mM neostigmine (2.6%).
Discussion
The current study aimed to investigate the substrate selectivity of acetylcholinesterase
and butyrylcholinesterase and also to study the inhibitory effects of various short, medium
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and long acting cholinesterase inhibitors. The results supported our first hypothesis that
certain substrates would be more selective for either cholinesterase and that this would be
visualized through higher relative velocity of colour change. Our second hypothesis that
longer acting inhibitors would result in subsequently lower RV was also partially supported.
In the current study, the RV of acetylcholine was considerably higher in the presence
of acetylcholinesterase (100%) compared to the presence of butyrylcholinesterase (33.8%)
(Figures 1, 2). Furthermore, butyrylcholine with acetylcholinesterase returned a RV of 2.6%
compared to 100% in butyrylcholinesterase (Figures 1, 2). These results agree with a
previous study by Çokugras (2003) in which acetylcholinesterase and butyrylcholinesterase
hydrolysed acetylcholine and butyrylcholine respectively most rapidly compared to other
substrates.
This enzyme specificity for certain substrates is due to the active site of
butyrylcholinesterase favouring longer alpha carbon chains and functional groups and the
active site of acetylcholinesterase favouring smaller groups (Saxena et al., 1997).
For the same reasons, it was expected that benzoylcholine would be hydrolysed more
rapidly by butyrylcholinesterase than acetylcholinesterase due to the large benzene ring on
the alpha carbon of this substrate. The current study confirmed this, with an RV of 104% in
butyrylcholinesterase and 1.1% in acetylcholinesterase (Figure 1, 2). It should, however, be
noted that benzoylcholine should still be hydrolysed slower than the control substrate
butyrylcholine due to a lower affinity to the binding site as described by Fraser (1956). It is
likely that inaccurate subjective human perception of colour change affected the result.
Similarly, suxamethonium is known to be hydrolysed by butyrylcholinesterase but not
Acetylcholinesterase due to the deep gorge in the active site within Butyrylcholinesterase
(Saxena et al., 1997). However, experimentally, it was observed that suxamethonium was not
hydrolysed by either cholinesterase. This discrepancy could be due to genetic variations in
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the Butyrylcholinesterase gene that translated to mutations in the binding site as described by
Boeck et al. (2002).
Similar mutations could explain the high RV of methacholine in the presence of
acetylcholinesterase (195%, Figure 1), which was unexpected. Indeed methacholine should
not be rapidly hydrolysed by acetylcholinesterase due to the steric hindrance caused by the
proximity of the methyl group to the ester group resulting in a lower rate of hydrolysis
(Bruning et al., 1996; Foye et al., 2008). For the same reason, methacholine should also resist
hydrolysis by Butyrylcholinesterase. This result was observed (RV=1.3%, Figure 2).
The final substrate, carbachol, was not hydrolysed by Acetylcholinesterase (Figure 1)
but slowly hydrolysed by Butyrylcholinesterase (RV=22%)(Figure 2). Rosenberry et al.
(2008) obtained similar results, and found that the hydrolysis of the carbamolyated enzyme
intermediate after the initial enzyme-carbachol complex is rate limiting. The slow hydrolysis
was expected and confirms that carbachol is not specific for either cholinesterase.
Overall the results showed that neither enzyme was more active than the other,
however, it was demonstrated that substrates with larger and smaller functional groups were
more rapidly hydrolysed by Butyrylcholinesterase and Acetylcholinesterase respectively.
The second part of the experiment, revealed the inhibitory actions of various
compounds. The addition of edrophonium significantly inhibited Acetylcholinesterase
(RV=25%, Figure 3) compared to Butyrylcholinesterase (RV=89.3%, Figure 4). Given that
edrophonium is a selective, short-acting acetylcholinesterase inhibitor, this was expected
(Cook, 1992). The medium acting cholinesterase inhibitors physostigmine and neostigmine,
inhibited Acetylcholinesterase, with RVs of 7.4% and 10.5% respectively (Figure 3).
Butyrylcholinesterase was inhibited by physostigmine (5.68%) and neostigmine (2.6%) to
similar degrees (Figure 4).
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It has been previously demonstrated that stigmines exert a greater inhibition compared
to shorter acting inhibitors such as edrophonium (Sakuma et al., 1992) Edrophonium, binds
electrostatically to the anionic site of Acetylcholinesterase and to the esteric site by hydrogen
bonding. The stigmines also bind electrostatically to cholinesterases, but produce longer
inhibition due to the formation of a strongly bound covalent carbamylated intermediate at the
esteric site (Riviere and Papich, 2009). This intermediate resists hydrolysis. Thus, even
though initial inhibitor concentration of the stigmines (5uM) was 16 times less than
edrophonium (80uM) the effect was greater. This confirmed part of our second hypothesis.
Furthermore, it can be seen that even at low concentrations (5-80uM), inhibitors can still
outcompete substrates at greater concentrations (100mM) for the cholinesterase binding site.
Malathion, as a long acting irreversible non-selective cholinesterase inhibitor (Dale et
al., 2007) would be expected to produce greater inhibition than edrophonium and the
stigmines. However the RV was unexpectedly accelerated in both Acetylcholinesterase
(RV=139.3%, Figure 3) and Butyrylcholinesterase (RV=113.6%, Figure 4) conditions,
disagreeing with the second hypothesis. An explanation could be that the malathion
concentration (50uM) was insufficiently high to inhibit cholinesterase. Indeed, a study by Pei
et al. (2010) revealed that the IC50 of malathion acting on ACETYLCHOLINESTERASE
was 10 and 1000 fold greater than edrophonium and neostigmine respectively.
The effects of atropine accelerated acetylcholinesterase (RV=121.9%, Figure 3) but
inhibited butyrylcholinesterase (RV=89.3%, Figure 4). The noted acceleration of
acetylcholinesterase by atropine is a well documented phenomenon. Indeed, Kato et al.
(1971) demonstrated this effect, showing that the deacetylation step in cholinesterase
hydrolysis would be aided by atropine. However, atropine is not known to interact with
Butyrylcholinesterase and the slight inhibition could be attributed to non-standardized
laboratory conditions. These same changes likely contributed in part to the unexpected results
8
of carbachol, which significantly inhibited both Acetylcholinesterase (17.1%, Figure 3) and
Butyrylcholinesterase (9.77%, Figure 4). A more likely explanation is that the high
concentration of carbachol (10mM) was sufficiently large to out-compete the substrate
acetylcholine (100mM) for cholinesterase and therefore inhibit the reaction.
The experiment had several problems. Firstly it was assumed that human detection of
visual change is accurate. Given some unexpected results, the use of a spectrophotometer is
recommended to improve accuracy in the future. Furthermore, only one concentration of
each substrate and inhibitor was used. Future direction points towards utilising a range of
physiologically appropriate concentrations and increasing the number of trials to improve
both the reliability and clinical applicability of the results. Laboratory conditions can also be
standardised with the use of an incubator.
In conclusion the aims of the study were satisfied. The selectivity of
acetylcholinesterase and butyrylcholinesterase for different substrates was demonstrated.
Furthermore, the action of both short and medium acting inhibitors was clearly elucidated.
However, the hypotheses were only partially supported, with malathion returning unexpected
responses. Clear flaws in methodology may rectify these problems, and future direction
points towards a stricter protocol.
Word Count: 1499
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