What is the role of serine and histidine in the mechanistic step shown in Figure 1? Propose a role for the catalytic triad glutamate residue.
Requirements: short answer
From Poison to Medicine
Mechanisms and an Antidote
Acetylcholinesterase catalyzes the hydrolysis of acetylcholine to acetic acid and choline. This is a two-stage process involving hydrolysis (Figure 1).
Figure 1: Acetylcholinesterase overall reaction
The first step forms the intermediate that is shown in the PDB ID: 2ACE crystal structure and is diagrammed in Figure 2 below. This covalent adduct is called a tetrahedral intermediate, because the trigonal planar carbonyl group in the acetylcholine forms a tetrahedral oxyanion upon nucleophilic attack by the serine oxygen:
Figure 2: Formation of the first tetrahedral intermediate in the AChE mechanism
Question 1: What is the role of serine and histidine in the mechanistic step shown in Figure 1? Propose a role for the catalytic triad glutamate residue.
The tetrahedral intermediate is fleeting. The electrons from the oxyanion push back down, reforming a double bond and releasing the choline product from the active site (Figure 3).
Figure 3: Release of choline in the AChE mechanism
An enzyme must return to its original state to catalyze another reaction. To perform this step, a molecule of water in the active site attacks the covalently bound acetate, forming a second tetrahedral intermediate (Figure 4). Like the first tetrahedral intermediate, this collapses, releasing the acetate product.
Figure 4: Formation of the second tetrahedral intermediate and acetate release
Question 2: Why is the hydrolysis step necessary? If water couldn’t get into the active site for this hydrolysis step, what do you predict the outcome would be?
Sarin is a powerful inhibitor of AChE. The structure of this poison is shown in Figure 5.
Figure 5: Sarin, a powerful nerve agent
An image of the active site residues interacting with sarin is provided in Figure 6. Examine the figure and answer the following questions.
Figure 6: Sarin bound to AChE
Question 3: Draw the mechanism that shows how serine reacts with the nerve agent sarin.
Question 4: What type of inhibition is this? Explain your answer. How does this lead to the symptoms you read about in the case study?
There are some chemicals that show promise as antidotes to sarin poisoning. HI-6 (Figure 7) is one such agent.
Figure 7: HI-6, a potential sarin antidote
If administered quickly, this compound can reverse the effects of sarin poisoning. Figure 8 shows HI-6 bound to sarin-inactivated acetylcholinesterase.
Figure 8: AChE bound to sarin and the potential antidote, HI-6
Question 5: Based on Figure 8, how do you think the antidote reacts to release sarin from the active site serine? Propose a structure of the antidote-sarin adduct, and show the mechanism of how this adduct forms, using arrows to show electron flow. You can represent the parts of the molecule that do not directly participate in the mechanism as R groups in your structure. The pKa of the antidote oxime functional group is 7.6.
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