Acetylcholinesterase is one of the most crucial enzymes for nerve response and function. Acetylcholinesterase (AChE) catalyzes the hydrolysis of acylcholinesters with a relative specificity for acetylcholine. The hydrolysis reaction proceeds by nucleophilic attack of the carbonyl carbon, acylating the enzyme and liberating choline. This is followed by a rapid hydrolysis of the acylated enzyme yielding acetic acid, and the restoration of the esteratic site. The pathway is similar to a pipeline, where the substrate goes in one end and the products come out the other through conformational changes, along with the hydrophobic and electrostatic forces. Acetylcholine is the neurotransmitter common to many synapses throughout mammalian nervous systerms. The enzyme is bound to cellular membranes of excitable tissues at cholinergic synaptic junctions, endoplasmic reticulum, among other membranes, which are usually associated with nerve impulse conduction. AChE is also found in red blood cells. The enzyme in its natural state is a monomer with a molecular weight around 60,000 and often forms aggregates which continue to produce catalytic activity, such as this image of a dimer. The enzyme monomer is an alpha/beta protein that contains 537 amino acids. It consists of a 12-stranded mixed beta sheet surrounded by 14 alpha helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The optimum pH for AChE is 7.0 with the isoionic point near the pH of 5.35. The specificity at substrate concentrations of 1 X 10-3M and constant enzyme level (approximately optimal for acetylcholine), produce the following relative rates of hydrolysis for several ester substrates: acetylcholine, 100; propionylcholine, 96; butyrylcholine, negligible; and triacetin, 11. A typical activator for AChE is 0.02 M Mg2+, which is stimulatory in purified preparations. The enzyme is stable as a lyophilized powder for months at -20deg.C and in solution at pH 7.0 for several days at 4deg.C.
The recent rendition of the x-ray structure for AChE places the active catalytic site deep within a gorge-like fold of the protein. Electrostatic computations reveal the enzyme to be a single massive dipole. Electrostatic potential maps quite seductively suggest that AChE, possibly like other enzymes with charged substrates, steers its substrate toward its gorge and into the active site. In this image the red represents negative proximities and the blue represents positive proximities. The following acetylcholinesterase movie shows the electostatic force in action through a 3-Dimensional view. The next movie shows the fluctuations in the dimensions of the active site gorge over a 60ps time period through conformational changes in the presence of model substrates.
Ramachandran Plot of Acetylcholinesterase shows the secondary structure distribution and frequency of the monomer protein. From this we see a variety of secondary structure with mostly alpha helices and beta sheets.
Use the left mouse button to manipulate the screen
To better view the hydrophobic/hydrophilic areas follow these instructions:
Click on each button in sequence from top to bottom or any combination desired.
Then right click mouse, hold and drag to select, then protein, then hydrophobic. Then right click again, hold and drag to color, select chain. Depending on the colors of your screen, the hydophobic areas of the beta sheets and helices should be blue and the hydrophilic areas should be red for helices and white for beta sheets.
You will then be able to see the unique pattern of hydrophilic residues on the outer surface and in the gorge, which is crucial for the enzyme to function properly. This will be described in the next section. The most prominent secondary structure is the large twisted beta sheet that forms a saddle shape incorporated with main beta hairpin turns. The alpha helices seem to be scattered throughout the structure without and key motifs.
The active site lies near the bottom of a deep and narrow gorge that reaches through the protein. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. The residues of the inner surface of active sight form regions that are charged and uncharge for specific binding orientation. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.There are also reports on a non-catalytic ligand binding-site remote from the active site, which can be seen in the binding and inhibition by fasciculin II.
The following are active site enlargements of the bottom of the gorge. The spherical model shows the amino acid side chains that interact with the inhibitor. To view aromatic and aliphatic residues in the active site follow these instructions:
First left click with the mouse on one of the active site enlargement buttons.
Then right click mouse, hold and drag to select, then protein, then aromatic and/or aliphatic. Then right click again, hold and drag to color, select chain. You can also choose select, then highlight selection. Depending on the colors of your screen, the aromatic and aliphatic areas of the active site should be shown in blue.
This will allow you to see the tremendous concentration of aromatic and aliphatic residues in the active site, which are rudimentary for the binding of the substrate.
Inhibition
Decamethonium is an inhibitor of AChe. In the decamethonium complex, the inhibitor lies parallel to the surface of the gorge, showing the depth and shape of the gorge.
In a complex with edrophonium, the quaternary nitrogen of the ligand interacts with the indole of Trp-84, and its m-hydroxyl displays bifurcated hydrogen bonding to two members of the catalytic triad, Ser-200 and His-440.
In a complex with tacrine, the acridine is stacked against the indole of Trp-84. The structure of tacrine with its 3 rings aligns with the aromatic residues in the active site.
Structure of acetylcholinesterase complexed with the nootropic alkaloid, huperzine A, which is found in an extract from a club moss that has been used for centuries in Chinese folk medicine. The affinities of structural huperzine A are correlated with their interactions with the protein, showing the importance of individual hydrophobic interactions between huperzine A and aromatic residues in the active-site gorge of AChE. Huperzine A and Tacrine are often used in the treatment of Alzheimer's disease.
Acetylcholinesterase inhibition by fasciculin, a protein found in snake venom, reveals a synergistic three-point anchorage consistent with the picomolar dissociation constant of the complex. Loop II of fasciculin contains a cluster of hydrophobic residues that interact with the peripheral anionic site of the enzyme and sterically occlude substrate access to the catalytic site. Loop I fits in a crevice near the lip of the gorge to maximize the surface area of contact of loop II at the gorge entry. The fasciculin core surrounds a protruding loop on the enzyme surface and stabilizes the whole assembly. Upon binding of fasciculin, subtle structural rearrangements of AChE occur that could explain the observed residual catalytic activity of the fasciculin-enzyme complex, located on the side of the protein. The AChE and fasciculin conformations in the complex are very similar to those in their isolated structures. Fasciculin is bound at the peripheral anionic site of AChE, almost sealing the narrow gorge leading to the active site, with the dipole moments of the two molecules roughly aligned. The high affinity of fasciculin for AChE is due to a remarkable surface complementarity, obviously due to evolutionary development.
As we can see, from the visual aids, the AChE enzyme can be inhibited by many different molecules with different binding sites. Hopefully this web page exploration of Acetylcholinesterase has given you a better understanding of this enzyme and proteins in general.
References
http://www.worthington-biochem.com/manual/C/ECH.html
http://bj.portlandpress.co.uk/bj/157/bj1570069.htm
http://chemcca10.ucsd.edu/~jmbriggs/ache.html
http://www.ls.huji.ac.il/~mirtag/home.html
http://www.weizmann.ac.il/~cskurt/che.html
http://bioinfo.weizmann.ac.il/_ls/israel_silman/israel_silman.html
Protein location: http://www.rcsb.org/pdb/
Special thanks to Dr. Jakubowski, CSB/SJU Chemistry Department, for providing html files.
