Recombinant Human Acetylcholinesterase: The Mental Breakdown
Created by Brian Au
Recombinant human acetylcholinesterase, (pdb ID = 3LII) from the species Homo sapiens, is an enzyme that catalyzes the breakdown of the neurotransmitter acetylcholine by rapid hydrolysis. Acetylcholinesterase (AChE for short) is a fast-acting enzyme which functions at a rate comparable to a diffusion-controlled reaction. The hydrolysis of acetylcholine by AChE plays an important role in neuronal apoptosis by terminating neurotransmission between the cholinergic synapses (1). By cleaving the acetylcholine into a choline and acetate, the function of acetylcholine is destroyed (3). Because recombinant human acetylcholinesterase (rhAChE for short) plays a role in controlling the signal transmission between neurons, there are studies being conducted utilizing acetylcholinesterase inhibitors to treat various neurological disorders. Because many forms of dementia, including Alzheimer’s disease, result from the reduction in the activity or death of cholinergic neurons, acetylcholinesterase inhibitors have been employed in studies to reduce the rate at which acetylcholine is broken down in the brain (1).
The molecular weight of one subunit of rhAChE is 67,796 Da and its isoelectric point (pI) is 5.87 (7). The two subunits of rhAChE are identical and combine to form a homodimer. The total molecular weight of the rhAChE dimer is 135,792 Da. There are 556 residues per subunit determined by the rhAChE crystal structure and a total of 1112 residues for the rhAChE dimer (1).
Recombinant human acetylcholinesterase’s function as an enzyme is directly related to the various structures that constitute a protein. The primary structure of rhAChE determines the folding that occurs in the protein itself as well as its mechanism. The primary structure of rhAChE shows a high amount of aromatic residues along with 63 positively and 53 negatively charged residues per subunit (4). The aromatic residues are an integral part of the gorge lining and help in determining the secondary and tertiary structures. The secondary and tertiary structures help in determining the shape and three-dimensional structures of each subunit of rhAChE and both play a role in determining the function of the enzyme. Recombinant human acetylcholinesterase is a globular protein and is ellipsoid in shape with dimensions of 45 x 60 x 65Å (1). The secondary structure of the rhAChE homodimer contains 12 β-sheets, 14 α-helices and a total of 74 random coils. A four-helix bundle composed of helices αF’3 and αH from each rhAChE subunit hold together the homodimer (6).
The tertiary structure of recombinant human acetylcholinesterase shows the α/β hydrolase fold which is common in many hydrolytic enzymes and is a defining feature for many proteins in the lipase, carboxylesterase and esterase super family. The α/β hydrolase fold is a tertiary fold that occurs in many proteins with no apparent sequence similarity. The fold consists of mostly parallel, eight-stranded β-sheet surrounded by α-helices which creates a “stable scaffold” for the active-site (4).
The three-dimensional structure rhAChE consists of two subunits, denoted A and B, combined by two disulfide bonds from the cysteine residues to form the protein dimer. The disulfide linkage was determined after SDS-PAGE revealed a 130 kDa band and is formed by the cysteine residues on each subunit. The rhAChE dimer consists of two crystallographically-independent copies of the acetylcholinesterase catalytic subunit. Further gel analysis shows that after cleaving the disulfide bonds between the dimers, there was a single intense 65 kDa band. The single band confirms that the two AChE subunits are nearly identical. The two subunits reduce the hydrophobic residues exposed on the surface of the protein by inducing folding. They also provide structure to the protein and create the active-site which facilitates the intake of substrates including acetylcholine and water (1).
With the combination of the 2 AChE subunits, a gorge is formed that contains the active-site of the rhAChE. The active-site gorge is a remarkable structure that demonstrates how primary, secondary and tertiary structures lead to the enzymatic function of rhAChE. From the surface of rhAChE, a narrow gorge 20Å in length leads to the catalytic triad 4Å from the bottom shown in fig. 1. The active-site gorge is lined with 14 aromatic residues which help to form 40% of the surface and the shape of the gorge. Demonstrating how the primary structure coincides with the function of a protein, aromatic rings lining the walls of the gorge help guide substrates to the active-site. These residues are highly conserved and appear in other forms of AChEs. One should note that the walls of the gorge contain only a few acidic residues including Asp-304 and Glu-285 at the top, Asp-74 hydrogen bonded to Tyr-337 in the middle and Glu-202 near the base. Residues which help facilitate the movement of the substrate (Ser-203, Asp-304, Glu-285, Asp-74, Tyr-337, Glu-202, Trp-286 and Tyr-70) are all coded for and synthesized in the first exon which codes residues 1-480. Aromatic residues such as Tyr-119 and Phe-338 help guide the substrate at the bottleneck and Trp-86 and Phe-338 are involved in helping guide the substrate into the active center (1).
The rate of ligand binding is extremely high therefore leading to high catalytic activity. To account for the high catalytic activity mechanisms have been proposed that take into account the aromatic components as well as the electrostatic characteristics of the gorge. One mechanism proposes that a dipole moment is created by the acidic residues (Asp-74, Glu-202, Glu-450) allowing the positively charged acetylcholine to move down the gorge. Mutations of Asp-74 showed a decrease in the bimolecular rate constant by 15 to 20-fold. Another mechanism utilizes the overall aromatic character of the gorge. The gorge is hydrophobic which results in a low local dielectric constant. A low dielectric constant produces a higher effective local charge than would be predicted from the small number of acidic group lining the gorge. The aromatic lining provides a mechanism where the aromatic rings provide low-affinity binding-sites that absorb the acetylcholine quickly and favorably energetically. The low hydration state of acetylcholine allows it to favor the pi-cation interaction with aromatic residues especially with Trp-286 and Tyr-70. The high hydration states of metals prevent them from entering and moving into the gorge. The low hydration states then can be subsequently released down to the active-site. The same mechanism could also be applied to the removal of the choline product (1).
Scientists have concluded that the active-site of rhAChE consists of two main subsites, the esteratic and anionic subsites. The esteratic subsite is the catalytic subsite of serine hydrolases, and the anionic subsite is responsible for interacting with the charged quaternary group of the choline moiety of acetylcholine (5). The anionic site contains Trp-86 and 6 to 9 COO- ions which bind the positively charged choline moiety of the acetylcholine using a combination of van der Waals attractions and electrostatic guidance of the negative charges. The electrostatic guidance mechanism proposes that an array of positive charges is able to navigate the negatively charged superoxide radical into the active-site. The esteratic site contains the three functionally important and highly conserved residues named the “catalytic triad” (1). The triad consists of Glu-334, Ser-203, and His-447 which form the planar active-site of acetylcholinesterase. The active-site is 4Å from the base of the gorge. The esteratic active-site with Ser-203 directly binds to the acyl group of the acetylcholine using a tetrahedral bond. Ser-203 contains a reactive hydroxyl group which attacks the substrate forming a covalent acyl-enzyme complex which rapidly deacylates (2). The serine, positioned after β5 strand, acts as the nucleophile in the “nucleophile elbow”, a structure which allows the substrate and the water molecule easy access to the site. The nucleophile elbow also contributes to the formation of the oxyanion-binding site which stabilizes the negatively charged transition state that occurs during the hydrolysis of the acetylcholine (4).
The oxyanion hole is a structure formed by the two backbone nitrogen atoms of the residues adjacent to the serine nucleophile and the imidazole group of His-447 which has been linked to reacting with the acetylcholine carbonyl oxygen. It has been suggested that a highly conserved chain of 10 residues including three glycine residues in a row help to form the oxyanion hole. Gly-120, Gly-121 and Ala-204 are the proposed residues that contribute to the formation of the oxyanion hole. The flexibility and small size of the glycine residues allow the chain enough flexibility to allow the amide nitrogens of both Gly-120 and Gly-121 to be a part of the oxyanion hole (1).
Both the oxyanion hole and the acyl pocket are responsible for substrate specificity and will only allow the substrate to be positioned in one way to undergo hydrolysis at the active-site. This specificity also prevents the binding of the abundant metal ions around the synaptic cleft to the active center. The acidic residue, glutamic acid, is located after the β7 strand, and the histidine residue is located after the last β strand (4). The protein has been also shown to have additional binding sites for acetylcholine and other quaternary ligands including the PAS, peripheral anionic binding site, and the ACS, aromatic cation binding site. One should note that the PAS is located 15Å from the active-site placing it at the mouth of the gorge and contains peptide sequences from residues 251-264 and 270-278. A fluorescent probe, propidium, has been used by scientists to determine that the PAS can be occupied by competitive inhibitors. The PAS has been shown to be involved in substrate inhibition of AChE which allows it to be a determining factor for the overall kinetics of the reaction. Elongated ligands (bisquaternary compounds) were able to blockade the gorge between the side chains of Trp-286 and Phe-338 as well as span from the PAS all the way down to one of the aromatic residues lining the gorge. The presence of a ligand at this position blocks the entry of substrates as well as the leaving of products from the hydrolysis. At a high enough concentration, the substrate acetylthiocholine was bound transiently to the PAS giving rise to substrate inhibition. At a low concentration the substrate binding to the PAS would accelerate the acylation step during hydrolysis (1).
Recombinant human acetylcholinesterase contains two associated ligands which contribute to the overall function of the protein. N-acetyl-D-glucosamine promotes communication between the PAS of the rhAChE and external cells. NAG interacts with the positively charged loop of an adjacent monomer within 4.5Å (fig. 2). Sulfate ions also are ligated to the surface of rhAChE which allow the protein to behave like an anion-exchange resin. Sulfate ions are also utilized in the crystallization process of rhAChE (1).
Because acetylcholinesterase hydrolyzes acetylcholine rapidly, there are substrates that can be used to inhibit the function of acetylcholinesterase by blocking the active-site. These substrates include carbamyls and phosphoryl esters which dissociate more slowly than the choline esters (2). This provides a mechanism that will inhibit the activity of rhAChE by slowing down the rate at which acetylcholine can be processed and hydrolyzed. Organophosphorus poisons are powerful toxins that are irreversible inhibitors of rhAChE because of their ability to form a covalent bond with Ser-203 (6). There are toxins that can complex with rhAChE including snake venom toxin fasciculin-II (1B41). Many drugs that combat neuromuscular diseases are aimed to inhibit the acetylcholinesterase by binding to the active-site and changing the overall conformation of the rhAChE. Mutations of rhAChE include mutant E202Q of rhAChE (pdb ID = 1F8U) (1). Mutant E202Q contains a “three-finger” polypeptide toxin purified from the venom of the Dendroaspis angusticeps. The toxin alters the acetylcholinesterase function by removing a charged group, Glu-202, from the protein core and substituting it with a neutral isosteric moiety. The residue Glu-202 is responsible for the hydrogen-bond network including Glu-450 and Tyr-133 which bridge molecules of water (10).
Recombinant human acetylcholinesterase shows an approximate similarity in the primary structure to many different proteins including those in the type-B carboxylesterase/lipase family (3). Recombinant human acetylcholinesterase belongs to the α/β hydrolase fold super family and will therefore show conserved segments of the primary, secondary, and tertiary structure (6). The protein PSI-BLAST search shows that rhAChE has both primary similarities to the catalytic domain of human bile salt activated lipase (pdb ID = 1F6W) and truncated recombinant human bile salt stimulated lipase (pdb ID = 1JMY). The E values were 3e-85 and 1e-82 respectively which means that the sequence similarity is high for both of these lipases in comparison to rhAChE.
PSI-BLAST shows that rhAChE has a high sequence similarity with the crystal structure of the synaptic protein neuroligin 4 (pdb ID = 3BE8) (E value = 2e-105). Neuroligin 4 and rhAChE both have the N-acetyl-D-glucosamine (NAG) ligand used in extracellular communication and are found in the synaptic junctions in the brain. The neuroligin 4 extracellular domain exhibits 32%-36% sequence identity and shares the same globular shape as acetylcholinesterase. Neuroligin 4 also shares the same α/β hydrolase fold which conserves the basic structure in the catalytic active-site; however the Ser-200 residue is replaced by a Gly-254 to disable the catalytic function. One noticeable difference shows that neuroligin 4 has three intra-molecular disulfide bridges, one which is unique to neuroligins, compared to the two disulfide bridges found in rhAChE (6).
With the use of the Dali structural alignment test, the tertiary structure of recombinant human acetylcholinesterase was compared to other structures in hope of finding proteins with similar tertiary structures. The structure of the catalytic domain of human bile salt activated lipase had a Z-score of 48.7 (rmsd = 2.5). The crystal structure of the synaptic protein neuroligin 4 had a Z-score of 47.6 (rmsd = 2.3), and the truncated recombinant human bile salt stimulated lipase had a Z-score of 49.3 (rmsd = 2.5) (9). The lipases were similar in the tertiary structure because they belong in the same family of α/β hydrolase folds and contain a functionally and structurally similar active-site (histidine residues conserved along with an acidic residue and nucleophile) and a similar β sheet structure.
Neuroligin 4 shares many tertiary structure features that are found in acetylcholinesterases. A Dali Lite structural comparison test compared neuroligin 4 with rhAChE and found a sequence identity of 39% (8). The neuroligin has similar size and shape with dimensions of 45 x 60 x 65Å and the same overall structure with 12 stranded central β sheet surrounded by 14 α helices. Both the neuroligin and AChE have at least two intra-molecular disulfide bridges, with neuroligin having an extra disulfide linkage unique to its family (6).
Both neuroligin and acetylcholinesterase are made up of two subunits related by a two-fold symmetry and linked through a tightly packed four-helix bundle made of helix α37,8 and the C-terminal helix α 10 from each subunit. When the neuroligin and AChE subunits combine, hydrophobic residues are covered with neuroligin 4 dimer burying 100% of the 875Å2 interface and AChE burying 77% of the 870Å2 interface. The burying of hydrophobic residues through the combination of subunits increases the stability of the protein dimer and in this case shows that the neuroligin is more stable than the AChE. Both proteins also share a central gorge with an active center. The topology and many of the lining residues of the gorge are well conserved between the two proteins. The only deviations which deactivate the active-site are found in the vestigial triad and the oxyanion hole residues (6).
Recombinant human acetylcholinesterase is an important protein in regulating neurotransmission in human beings. Understanding the structure of the enzyme will illuminate the intricacies of the mechanisms and functions that rhAChE operates. Understanding how the primary, secondary, and tertiary structures work together as pieces of a whole instead of separate entities will allow humans to modify the enzyme to help in the battle with neuromuscular diseases and the many forms of dementia.