Acetylcholinesterase and Butyrylcholinesterase Substrate Selectivity and Various Acting Cholinesterase Inhibitors

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Acetylcholinesterase and Butyrylcholinesterase substrate selectivity and various acting cholinesterase inhibitors Introduction
Cholinesterases are a group of enzymes present in mammals which breakdown certain neurotransmitters by hydrolyzing the ester bonds within a molecule (Rang & Dale, 2007). There are two major types of enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Though similar in structure, they differ in distribution, function and substrate specificity. AChE is found in red blood cells, cholinergic fibres and muscle (motor end-plate), existing as mainly membrane bound (Rang & Dale, 2007). It is highly specific for the neurotransmitter acetylcholine (ACh) and its principle role is termination of impulse transmission at cholinergic synapses through hydrolysis of ACh (Rang & Dale, 2007). Produced in the liver, BChE resides in the plasma and many tissues. Similarly, both enzymes hydrolyse ACh to choline and acetic acid with BChE hydrolysing BCh more rapidly than ACh (Rang & Dale., 2007). The substrate traffic in AChE involves two distinct binding sites, the catalytic and peripheral anionic sites, with BChE having relatively broader substrate specificity than AChE (Colletier et al., 2006). Anticholinesterases (AnAChE’s) inhibit cholinesterase activity and depending upon the nature of their interaction with the active site are categorised as either short/medium duration or irreversible (Rang & Dale, 2007). The study of cholinesterase hydrolysis and inhibitory actions demonstrate the key pharmacological principles of enzymatic catalysis and proteins as targets for drug action. Their importance is evident in a study conducted by Kumar et al (1989), where significantly lower cerebrospinal fluid AChE activity in early onset patients of Alzheimer’s disease and an altered AChE/BChE ratio, lead to elevated levels of choline. The present study has two primary aims. The first is to demonstrate the substrate selectivity of AChE and BChE in the presence of a variety of cholinergic agents, including acetylcholine, butylcholine, methacholine, carbachol and suxamethonium. The second is to test the inhibiting action of differing cholinesterase inhibitors including physostigmine, neostigmine, malathion, edrophonium, atropine and carbachol. Enzyme activity will be measured as a function of velocity of the colour change of a red alkaline Tris-buffer to an acidic yellow relying on the principle of acid liberation upon substrate hydrolysis. It was hypothesised that BChE will exhibit broader substrate specificity compared to AChE. Furthermore, when in the presence of differing acting cholinesterase inhibitors, the action of AChE and BChE will be reduced to differing degrees. Results:

Figure 1. The velocity of Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE) relative to the control sample* in the presence of different substrates (*Acetylcholine was reacted with AChE and Butyrylcholine was reacted with BChE as the control samples)

Figure 2. The velocity of Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE) relative to the control sample* in presence of different inhibitors (*Acetylcholine was reacted with AChE and Butyrylcholine was reacted with BChE as the control samples) AChE and BChE both hydrolysed ACh. However, BChE reduced the relative velocity by half compared with AChE (Figure 1). BCh and Benzoylcholine were hydrolysed by BChE only, whereas only AChE hydrolysed methacholine. Benzolycholine reacted significantly slower than BCh in BChE (Figure1). Methacholine only achieved half the velocity compared with ACh in AChE. Carbachol and Suxamethonium could not be hydrolysed by both AChE and BChE (Figure1). In general, Malathion and Atropine had minimal effect on AChE and BChE as the velocity was only approximately 10% slower than the control samples (Figure2). Edrophonium had minimal effect on BChE whilst velocity was significantly decreased in AChE (Figure2). Hydrolysis by AChE and...
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