The Effect of Inhibitors on Cholinesterases and its interaction with specific substrates
INTRODUCTION
Enzymes are catalytic units within the body that help to increase the rate at which metabolic processes occur, to allow for optimal functionality within the body (Paraonau and Layer, 2008). Neurotransmitters play a vital signaling component in mammalian systems, and can be categorized into various groups, according to their functions. The chemical interaction between cholinesterases, an enzyme group, and its hydrolysis of Acetylcholine, a neurotransmitter, can be seen below:
In the human body, two sub-types of cholinesterases can be found:
True cholinesterases (Acetylcholinesterase):
Acetylcholinesterases (AChE) are enzymes that play a vital role in neurotransmitter reuptake in cholinergic synapses within the central nervous system and in neuromuscular junctions This enzyme has a particularly high specificity for acetylcholine, and can degrade roughly 5000 molecules of the neurotransmitter every second (Arias et al, 2010). Acetylcholine is a vital neurotransmitter within the body that allows for depolarization of the nerve cell, leading to the effect of muscle contraction. AChE serves to degrade ACh in the synapse so as to prevent muscle fatigue, which can be detrimental to a person’s health (Gandevia et al, 1996).
Pseudocholinesterases/Serum cholinesterase (Butyrylcholinesterase):
Butyrylcholinesterases (BChE) is a more widely distributed neurotransmitter, playing roles in areas including but not limited to; plasma, brain, skin, and the liver (Cohen, 2006). Because of this, its specificity is not limited to acetycholine, giving it the ability to metabolise other chemicals, namely Benzoylcholine, and is clinically important in the detoxification of other drugs (Mack and Robitzki, 2000).
While catalysis of substrates is an important metabolic function, their inhibition is also a vital activity that must be observed and measured. Inhibition of enzyme activity can have both positive and negative impacts in the body; for example, as mentioned earlier, over stimulation of skeletal muscles can lead to fatigue, hence AChE activity must be controlled. Conversely, inhibition of AChE is actually a form of treatment in diseases such as Alzheimer’s Disease (Gauthier, 2012), as the patient requires more acetylcholine in their synaptic clefts for motor movement.
This experiment aims to measure the substrate specificities of the enzymes AChE and BChE, on Acetylcholine and other substrates, to gain a wider understanding of their activity throughout the body. Furthermore, the relative velocity of both enzymes in hydrolyzing AchE in the presence of inhibitors will be measured, so as to relatively quantify our understanding of how inhibitors, in different concentrations, can affect the reactions between enzyme and substrate.
METHOD
This experiment utilised the methodology outlined in pages 22 – 24 of the UTS 91707 Pharmacology Practical manual (Rodgers, 2015), with the following amendments: recording the reaction times as seconds rather than minutes, thus affecting the calculated values for our relative velocities within our results; and sampling triplets of 250ul aliquots from the incomplete reaction tubes into the ELISA plates, rather than 200uL.
RESULTS
Table 1 and Table 2 specifically compare bench #3’s experimental values of enzyme activity, to the average of values obtained by the whole cohort. Enzyme activity for AChE and BChE was initially recorded in seconds, then converted to relative velocity percentages by the following equations:
For samples that were able to reach completion within 60 minutes:
For samples that were unable to reach completion by 60 min:
To calculate the percentage changes of samples that did not reach completion:
