Recombinant human Acetylcholinesterase (PDB ID = 3LII)
Created by Michael Marotta
Acetylcholinesterase (AChE) is an incredibly fast-acting hydrolase enzyme that ends nerve signal transmission in cholinergic synapses through hydrolysis of acetylcholine into acetic acid and choline (Dvir, Silman, Harel, Rosenberry, and Sussman, 2010). It is a fairly large protein and "it is now generally agreed that solubilized acetylcholinesterase exists in multiple forms, ranging in molecular weight from 60000 to over 400000" (Levinson, Ellory, 1973). The isoelectric point for AChE falls within the range of 4.6 to 5.2 (Fischer, Ittah, Gorecki, and Werber, 1995). Studies focusing on AChE activity in the fruit fly, Drosophila Melanogaster, found the enzyme to be concentrated in the organism's head region, indicating its pivotal role in the central nervous system (Melanson, Chang, Pezzementi, and Pezzementi, 1984). Neuromuscular junctions are also sites of high AChE activity. This protein carries important practical significance, as inhibition of AChE plays a crucial part in developing effective nerve poisons, pesticides, and several medicines treating neurological diseases (Dvir et al., 2010).
The foremost use of AChE inhibition in medicine is in the development of drugs for treatment of Alzheimer disease. The focus of medical study is on the characteristic selective loss of the globular membrane-bound molecular form of AChE in Alzheimer tissue rather than the asymmetric, aqueous soluble forms also present (Ogane, Giacobini, Struble, 1992).
When AChE is compared to cell adhesion protein neuroligin-1 (PDB ID: 3BIX), the proteins exhibit 70% sequence similarity (Arac, Boucard, Ozkan, Strop, Newell, Sudhof, Brunger, 2007). The function of neuroligin-1, however, is involved in synapse specificity and connectivity between neurons (Arac et al., 2007). These two proteins are fascinating to compare against one another.
Although their functions vary somewhat despite high sequence similarity, they are both vitally important to neurological health. Several brain disorders, including autism and mental retardation, are linked to abnormalities in synapse specificity and development (Arac et al, Introduction, 2007).
The relationship between AChE's structure and its function as an enzyme is closely tied. The protein consists of 540 residues and is organized into two identical chains,
labeled A and B, which form a homeodimer (Dvir et al., 2010). The
secondary structure is fairly diverse, having 24 alpha- helices made up of 185 residues and 25 Beta-sheets consisting of 91 residues. There are several individual residues that play essential roles in how AChE interacts with certain substrates. For example, the
S200 residue, located at the active site, irreversibly inhibits the enzyme by forming a covalent bond with organophosphorus poisons (Dvir et al., 2010). The function of AChE is effectively blocked and severe poisoning can occur due to the buildup of acetylcholine (See AChE inhibiting drugs slide for inhibitor structural binding details).
The
active site of AChE is its characteristic feature and is fascinating in its complexity and uniqueness. The active site is defined by a
deep, narrow ravine leading approximately halfway into the enzyme. The active site "gorge" is made up of several parts or "subsites" (Dvir et al., 2010). The three main subsites are the peripheral, the anionic, and the esteratic. The structure of the
peripheral site, marked by W-279, is very much involved in the active site's function, especially in acetylcholine (ACh) hydrolysis. The binding of an ACh molecule to the peripheral site will not only block the exit of choline product, but also effectively produce substrate inhibition (Dvir et al., 2010). It also serves as the initial "trapping point" for substrate.
The anionic site is the binding pocket location for ACh, AChE's natural substrate (Dvir et al., 2010).The AChE enzyme hydrolyzes the ACh so rapidly that the rate starts approaching that of diffusion controlled reactions (Dvir et al., 2010). Electrostatic potential and a high number of aromatic sites are the driving forces behind the fast reaction. The electrostatic potential derives from the charge difference between the positive ACh and the negative binding site. The chasm's walls are lined with 14 highly conserved aromatic residues (Y-70, W-84, W-114, Y-121, Y-130, W-233, W-279, F-288, F-290, F-330, F-331, Y-334, W-432, Y-442) (Sussman, Harel, and Silman, 1993). One of these residues, W-84, plays an integral role at the binding site of acetylcholine by making near contact with the quaternary group of the choline moiety (Sussman et al., 1993). While the mechanism is not fully known,it has been proposed that the aromatic ring structures serve to guide the Ach molecule through absorption to low-affinity sites, where it can then diffuse to the active site (Dvir et al., 2010).
Located at the bottom of the ravine, the esteratic site is defined by a catalytic triad made up of three highly conserved residues; H-440, E-327, and the S-200 previously mentioned. The acetylcholine molecule is thought to span between the esteratic and anionic sites during binding.
Another contributor to AChE's incredible reaction rate is the oxanion hole located immediately next to the esteratic site. It is created through amide hydrogens directionally oriented into the active site from the G-118, G-119, and A-201 residues (Dvir etal., 2010). The oxanion hole acts a stabilizer to the substrate and therefore greatly increases the catalytic activity of AChE.
Surprisingly, the conformation of AChE shows very little flexibility for such a big enzyme (Dvir et al., 2010).One of the few conformational changes that does take place occurs in the active site with "movement of the acyl pocket in the conjugate of the anti-Alzheimer drug, rivastigmine,with TcAChE (Torpedo californica AChE)" (Dvir et al., 2010). A huge amount of interest is being directed to AChE inhibiting drugs like rivastigmine (Structure and details taken from Drosophila melanogaster AChE. Harel et al., 2000).
Rivastigmine serves to increase cholinergic activity in the central nervous system, possibly even slowing the progression of Alzheimer Disease (AD) (Sabbagh, Farlow,Relkin, and Beach, 2006). This would deviate from previous expectations of solely symptomatic treatment. Its inhibitory function prevents further breakdown of ACh, a neurotransmitter AD patients are usually deficient in.
This kind of progress in battling such a formidable brain disorder is truly exciting and is evidence that intense study of AChE's structure has extremely practical uses in various fields. Acetylcholinesterase is a fascinating enzyme and will certainly be the subject of much study in the years to come.