A comparison of the relative reaction velocities of acetylcholinesterase and butyrylcholinesterase hydrolysing different cholinergic substrates and acetylcholine in the presence of a range of inhibitors

Introduction
Acetylcholine (ACh) is part of the autonomic nervous system and is found at nerve endings in both the peripheral and central nervous system at neuromuscular junctions, all autonomic ganglia, and many synapses. i ACh is a neurotransmitter that is synthesized in certain neurons from acetyl coenzyme A and choline by the enzyme choline acetyltransferase as shown in Figure 1. ii Figure 1.iii
Cholinesterases are enzymes which catalyse the breakdown of cholinergic substrates; an example of this is the breakdown of ACh into acetic acid and choline through a hydrolysis reaction catalysed by acetylcholinesterase as shown in Figure 2. This allows cholinergic neurons to return to their pre-activation resting states. Cholinesterases contain an anionic site and an esteratic site. The cholinergic substrate, such as ACh, binds to the anionic site. The acetyl group is transferred to the esteratic site and a free choline molecule is thus released. The acetylated enzyme then undergoes hydrolysis which releases the enzyme and in the case of ACh, acetic acid.iv This process can be completed on a number of different cholinergic substrates by acetylcholinesterase and butyrylcholinesterase.

Figure 2.v
There are two types of cholinesterase in the human body, these are; acetylcholinesterase (AChE), known as true cholinesterase, and butyrylcholinesterase (BChE), known as pseudocholinesterase.vi AChE is involved in the rapid hydrolysis of ACh in cholinergic pathways in both the central and peripheral nervous systems. BChE is also involved in hydrolysis reactions. It is found mainly in the liver where its target cholinergic substrate is butyrylcholine. BChE can still hydrolyse ACh however it is much less efficient then AChE at doing so.vii
Inhibitors can reduce or remove the ability for cholinesterases to breakdown cholinergic substrates. Cholinesterase inhibitors work by actively competing with ACh, occupying and blocking the site where the cholinergic substrate binds to the cholinesterase.viii
The experiment was designed to examine the effectiveness of AChE and BChE in hydrolysing different substrates and the affect the introduction of inhibitors to the solution had on the hydrolysis of acetylcholine. A comparison was made by measuring the time it took each sample to be completely hydrolysed compared to a control solution of acetylcholine and AChE or BChE. It is expected that AChE will have a greater relative reaction velocity when hydrolysing ACh than any other substrate. The introduction of inhibitors should reduce the relative reaction velocity of the AChE and BChE hydrolysis of ACh.
Methods
The time it took AChE and BChE to completely hydrolyse different substrates was recorded. The time it took for AChE and BChE to hydrolyse ACh in the presence of different inhibitors was also measured. For solutions which failed to completely hydrolyse, an assay was undertaken to measure the percentage change in comparison to two control tubes, one containing acetylcholine and AChE the other acetylcholine and BChE.
The experiment was completed following the instructions found in the School of Medical and Molecular Biosciences, Autumn Semester 2015, Pharmacology 1 Course Manual, Practical 2 Cholinesterase and its inhibitors.
Changes to the procedure included; changing the time measurements from minutes to seconds and the amount of solution used for the assay was changed from 200 microlitres to 250 microlitres.
Results
AChE and BChE were found to have different relative reaction velocities depending on the substrate they were hydrolysing. Inhibitors were also found to