Acetylcholinesterase (PDB ID: 1OCE)
Created by Meredith Sharp
Acetylcholinesterase (AChE) is an enzyme that degrades the neurotransmitter acetylcholine (ACh) following its release from a cell, terminating synaptic transmission of a signal between neurons (5). AChE accomplishes this degradation by catalyzing the hydrolysis of ACh into acetate and choline (1). Current research suggests that AChE inhibitors may have therapeutic potential in treating Alzheimer’s disease, a common and debilitating neurodegenerative disorder (4).
Acetylcholinesterase (AChE) is a large, 61 kDa protein composed of 537 amino acids. It consists of only one polypeptide chain, which contains 26 alpha helices and 23 beta strands. The source of the crystal structure of carbamoylated AChE (PDB ID: 1OCE) is Torpedo californica, more commonly known as the Pacific electric ray (3).
Although it is classified generally as a hydrolase, AChE is more specifically part of the carboxylesterase family, which includes proteins that degrade carboxylic esters (3). One conserved domain of AChE is a catalytic triad of residues at the active site, composed of a serine, a glutamate or aspartate, and a histidine. AChE’s catalytic triad contains Ser, Glu, and His. In addition, the substrate binding pocket, an alpha/beta hydrolase fold, is a conserved sequence of carboxylesterases (6).
The function of AChE is linked closely to its structure. AChE is a serine protease, and the active site is located at the end of a narrow, 20-Å deep, substrate-binding pocket (1). Aromatic residues line the binding pocket, creating low-affinity binding sites that guide the substrate to the active site. There is an electrostatic dipole directed down the binding pocket, which attracts positively charged substrates, such as the quaternary amine moiety of ACh, to the active site (2). The active site consists of the aforementioned catalytic triad of residues: Glu327, His440, and Ser200, which cleave the substrate molecule (3). Serine acts as a nucleophile to attack the carbonyl carbon of its substrate, acetylcholine (ACh). Histidine functions as a general acid-base catalyst to increase the nucleophilicity of serine, and glutamate stabilizes the transition state through low-barrier hydrogen bonding (7).
There are two important anionic sites in AChE. Trp84 is a part of the catalytic anionic site, which is located at the active site. When acetylcholine binds, Trp84 interacts with the choline group (5). Trp279 is a critical residue of the peripheral anionic site, which lies at the entrance of the binding pocket and has a putative regulatory function (3).
Another notable feature of AChE is the oxyanion hole at the active site, which consists of three residues: Gly118, Gly119, and Ala201. These residues stabilize the oxyanion during the transition state by forming bonds between the carbonyl oxygen of the substrate and their own peptidic NH groups (7).
There are several known inhibitors of AChE that function via carbamoylation of Ser200. The process of carbamoylation involves the addition of a carbamate, such as the physostigmine analogue 8-(cis-2,6-dimethylmorpholino)octylcarbamoyleseroline (MF268). The carbamoylate-enzyme complex is significantly more stable than choline-enzyme complex, causing a dramatic reduction in the rate of hydrolysis. MF268 is a pseudo-irreversible inhibitor of AChE, meaning that although its binding is technically reversible, it binds to the active site with such high affinity that it effectively acts as an irreversible inhibitor.
Although AChE has been studied extensively, its mechanism is still under investigation. Of the known enzymes, AChE boasts one of the highest rates of catalysis, which approaches the rate of diffusion (5). Study of the carbamoylated enzyme may serve to elucidate its mechanism of hydrolysis. Carbamoylation of AChE freezes the enzyme in its transition state to allow visualization of this rapid step (3). MF268 is a carbamate containing a long, bulky alkyl chain. When MF268 binds to the active site of AChE, the eseroline group serves as the leaving group, while the carbamate group binds to Ser200. Crystallographic evidence indicates that although the alkyl chain of MF268 sterically blocks the gorge leading to the active site, the enzyme is able to clear the eseroline group. This data suggests the presence of a putative “back door” opening in the enzyme (3).
The enzyme's high reaction rate further supports the “back door” hypothesis. The gorge is only wide enough to accommodate one substrate molecule at a time, so the hydrolysis products would have to traverse the entire length of the gorge in order to exit the enzyme before another molecule could enter. Consequently, a mechanism in which choline exits the active site via the gorge would be in contrast with the enzyme’s high rate of catalysis. Furthermore, the electrostatic dipole that guides ACh into the active site would counter diffusion of the positively-charged choline product out of the active site.
Thus, because the long, narrow shape of the binding pocket is unfavorable for substrate release, a conformational change of the enzyme could achieve clearance of the substrate more feasibly (2). The most plausible “back door” opening involves a short channel leading from the active site to the surface of the enzyme, with Trp84 positioned at the entry and Glu445 positioned at the exit. However, this mechanism is still under investigation (1).
Carbamoylate derivatives may be useful as a therapeutic approach to Alzheimer’s disease (AD), a neurodegenerative disorder. Carbamate-based AChE inhibitors can strengthen the responses of existing neurons by extending the half-life of ACh in the synapse. This magnification of the effects of ACh works to counter neuronal loss sustained by AD sufferers (4). Thus, although acetylcholinesterase has been a subject of interest for many decades, study of its carbamoylated form has the potential to elucidate a method of AD drug therapy.