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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

195.0
200.0
180.0
160.0
140.0
120.0
100.0 100.0
100.0

80.0
60.0
40.0
Relative Velocity (%)
20.0
0.0 0.0 2.6 1.1 0.0 0.0
0.0
1 2 3 4 5 6 7 8 9

Condition

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

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water were individually added to acetylcholinesterase – condition (2) water; (3)

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.

120.0
104.2
100.0 100.0
100.0

80.0

60.0

40.0 33.8

21.9
20.0
Relative Velocity (%)
0.0 0.0 1.3 0.0
0.0
1 2 3 4 5 6 7 8 9
Condition

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)

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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 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.

160.0

139.3
140.0

121.9
120.0

100.0

80.0

60.0

Relative Velocity (%)


40.0
25.0
17.1
20.0
10.5
7.4

0.0
Physostigmine Neostigmine Edrophonium Malathion Carbachol
(10mM)

Inhibitor

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.

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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

was, however, decreased in 80mM edrophonium (25.0%), 10mM carbachol (17.1%), 5mM

neostigmine (10.5%), and 5mM physostigmine (7.4%)(Figure 3).

120.0 113.6

100.0
89.3 89.3

80.0

60.0

40.0

Relative Velocity (%)


20.0
9.8
5.7
2.6
0.0
Physostigmine Neostigmine Edrophonium Carbachol
(10mM)
Inhibitor

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%).

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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

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.

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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

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

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(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).

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.

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(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

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.

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