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


In the presence of water and Tris buffer.0 60. and malathion (irreversibly acting) on the hydrolysis of acetylcholine and butyrylcholine were observed (Dale et al.0 0. 2007). physostigmine and neostigmine (medium-acting).0 100.0 100.0 Relative Velocity (%) 20.inhibitors edrophonium (short-acting).6 1.0 2.0 120. 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.1 0.0 0.0 160.0 40. while in the remaining conditions various 100 mM substrates or 2 .0 80.0 0.0 180. The aims of this practical were to demonstrate the selectivity of acetylcholinesterase and butyrylcholinesterase for various cholinergic compounds. the RV was anticipated to decrease as the duration of action of the inhibitor specific for the cholinesterase in use increased. Atropine and carbachol action.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.0 100. a cholinergic antagonist and agonist respectively.0 0. in condition (1) 100 mM acetylcholine was added to water and mixed. were also investigated. and to examine the inhibitory action of various anticholinesterases.0 195. Results 200.0 140.. Furthermore.

0 0. condition 1 (acetylcholine with water) and condition 2 (water with acetylcholinesterase) did not produce any colour change.1%). (8) methacholine. (4) butyrylcholine.0 104. (5) butyrylcholine. 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. (3) acetylcholine. (4) acetylcholine. in condition (1) 100 mM Butyrylcholine was added to water and mixed. methacholine produced the fastest relative velocity of colour change in acetylcholinesterase (RV=195%). In the presence of water and Tris buffer. 120. In Figure 1. (6) benzoylcholine. (3) butyrylcholine. (8) methacholine. (7) carbachol. (5) acetylcholine.0 20.3 0. followed by acetylcholine (RV=100%). carbachol.0 40.0 Relative Velocity (%) 0.8 21. (9) suxamethonium.0 60.0 80. butyrylcholine (RV=2. suxamethonium.2 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. Furthermore. (6) benzoylcholine.9 100.water were individually added to acetylcholinesterase – condition (2) water. (9) 3 . Both the degree and time of colour change were recorded. (7) carbachol.6%) and benzoylcholine (R=1. while in the remaining conditions various 100 mM substrates or water were individually added to ButyrylcholineE – condition (2) water.0 100.0 0.0 100.0 33.0 1.

0 100.5 17. However. followed by butyrylcholine (100%). suxamethonium.0 139.9 Relative Velocity (%) 40. 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. and 100 mM acetylcholine was then added to this mixture.1 0.0 140.0 7.4 10. Various inhibitors were added to Tris buffer and Acetylcholinesterase. 160. Both the degree and time of colour change were recorded.0 80. condition 1 (butyrylcholine with water) and condition 2 (water with butyrylcholinesterase) did not produce any colour change. 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.0 60.9%) and methacholine (1.0 20.2%).suxamethonium.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. acetylcholine (33.0 120. In Figure 2. Both the degree and time of colour change were recorded. 4 . carbachol (21.0 25. benzoylcholine produced the fastest RV in butyrylcholinesterase (104.8%).3%).3 121.

1%). 10mM carbachol (9.The RV for the hydrolysis of acetylcholine by acetylcholinesterase (100%) was increased to 139.3% in the presence of 50mM malathion. Both the degree and time of colour change were recorded. Various inhibitors were added to Tris buffer and butyrylcholinesterase. 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. 5mM physostigmine (5.0 5. 120.4%)(Figure 3). decreased in the presence of 80mM edrophonium (89. and 5mM physostigmine (7.7 0.9% in 5mM atropine (Figure 3).0 113. the RV for the hydrolysis of butyrylcholine by butyrylcholinesterase (100%) was increased in the presence of 50mM malathion (113. It was.5%).0 60.6 Inhibitor Figure 4: Relative percentage velocity of colour change of butyrylcholine in butyrylcholinesterase in the presence of various inhibitors. however.8 2. 10mM carbachol (17. In Figure 4.3 89. It was. and 5mM neostigmine (2.3%).6 89.8%). however. decreased in 80mM edrophonium (25. and to 121.6%).0 100.0 80.3 Relative Velocity (%) 20. 5mM neostigmine (10.0 Physostigmine Neostigmine Edrophonium Carbachol (10mM) 9. 5 .3%).0%).0 40. and 100 mM butyrylcholine was then added to this mixture.6%). 5mM atropine (89.7%).

however. For the same reasons. It should. 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. 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.1% in acetylcholinesterase (Figure 1. 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. In the current study. Our second hypothesis that longer acting inhibitors would result in subsequently lower RV was also partially supported. 2). 1997).8%) (Figures 1. the RV of acetylcholine was considerably higher in the presence of acetylcholinesterase (100%) compared to the presence of butyrylcholinesterase (33. 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. The current study confirmed this. with an RV of 104% in butyrylcholinesterase and 1. 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. It is likely that inaccurate subjective human perception of colour change affected the result. 2). 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). medium and long acting cholinesterase inhibitors. 2).. butyrylcholine with acetylcholinesterase returned a RV of 2. Furthermore.6% compared to 100% in butyrylcholinesterase (Figures 1.

1997). and found that the hydrolysis of the carbamolyated enzyme intermediate after the initial enzyme-carbachol complex is rate limiting. revealed the inhibitory actions of various compounds. (2002). Foye et al. was not hydrolysed by Acetylcholinesterase (Figure 1) but slowly hydrolysed by Butyrylcholinesterase (RV=22%)(Figure 2). it was observed that suxamethonium was not hydrolysed by either cholinesterase. short-acting acetylcholinesterase inhibitor. This result was observed (RV=1. Figure 3) compared to Butyrylcholinesterase (RV=89. carbachol.. 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.3%. The slow hydrolysis was expected and confirms that carbachol is not specific for either cholinesterase. Figure 4). Rosenberry et al. Similar mutations could explain the high RV of methacholine in the presence of acetylcholinesterase (195%. Overall the results showed that neither enzyme was more active than the other.. 2008). 1996. 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. Given that edrophonium is a selective.. methacholine should also resist hydrolysis by Butyrylcholinesterase. experimentally. 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.3%.Similarly. this was expected 7 . The addition of edrophonium significantly inhibited Acetylcholinesterase (RV=25%. Figure 1). 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. The final substrate. (2008) obtained similar results. which was unexpected. For the same reason. however. Figure 2).

but produce longer inhibition due to the formation of a strongly bound covalent carbamylated intermediate at the esteric site (Riviere and Papich. a study by Pei et al. This intermediate resists hydrolysis. it can be seen that even at low concentrations (5-80uM).4% and 10. Kato et al. This confirmed part of our second hypothesis. binds electrostatically to the anionic site of Acetylcholinesterase and to the esteric site by hydrogen bonding.68%) and neostigmine (2.6%.3%. as a long acting irreversible non-selective cholinesterase inhibitor (Dale et al. However the RV was unexpectedly accelerated in both Acetylcholinesterase (RV=139.(Cook.5% respectively (Figure 3). It has been previously demonstrated that stigmines exert a greater inhibition compared to shorter acting inhibitors such as edrophonium (Sakuma et al. Furthermore.. disagreeing with the second hypothesis. Butyrylcholinesterase was inhibited by physostigmine (5. inhibitors can still outcompete substrates at greater concentrations (100mM) for the cholinesterase binding site. 1992). Indeed. Figure 4). even though initial inhibitor concentration of the stigmines (5uM) was 16 times less than edrophonium (80uM) the effect was greater.3%. Figure 3) but inhibited butyrylcholinesterase (RV=89. Indeed. 2007) would be expected to produce greater inhibition than edrophonium and the stigmines. inhibited Acetylcholinesterase. The noted acceleration of acetylcholinesterase by atropine is a well documented phenomenon. Malathion.. The medium acting cholinesterase inhibitors physostigmine and neostigmine. 1992) Edrophonium. Figure 4) conditions. Figure 3) and Butyrylcholinesterase (RV=113. (2010) revealed that the IC50 of malathion acting on ACETYLCHOLINESTERASE was 10 and 1000 fold greater than edrophonium and neostigmine respectively. 8 . with RVs of 7. An explanation could be that the malathion concentration (50uM) was insufficiently high to inhibit cholinesterase. Thus.6%) to similar degrees (Figure 4). The effects of atropine accelerated acetylcholinesterase (RV=121. The stigmines also bind electrostatically to cholinesterases. 2009).9%.

Figure 3) and Butyrylcholinesterase (9. These same changes likely contributed in part to the unexpected results of carbachol. which significantly inhibited both Acetylcholinesterase (17. Laboratory conditions can also be standardised with the use of an incubator. Given some unexpected results. the action of both short and medium acting inhibitors was clearly elucidated. 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. showing that the deacetylation step in cholinesterase hydrolysis would be aided by atropine. the hypotheses were only partially supported. Furthermore. However. with malathion returning unexpected responses. Firstly it was assumed that human detection of visual change is accurate. only one concentration of each substrate and inhibitor was used. However. Word Count: 1499 9 .77%. and future direction points towards a stricter protocol.1%. the use of a spectrophotometer is recommended to improve accuracy in the future. The selectivity of acetylcholinesterase and butyrylcholinesterase for different substrates was demonstrated. atropine is not known to interact with Butyrylcholinesterase and the slight inhibition could be attributed to non-standardized laboratory conditions. Figure 4). Clear flaws in methodology may rectify these problems. In conclusion the aims of the study were satisfied. Furthermore.(1971) demonstrated this effect. The experiment had several problems. 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.

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