Human Choline Acetyltransferase (PDB ID: 2FY3)
Created by Chen Lin
Choline acetyltransferase is the enzyme responsible for catalyzing the reversible synthesis of acetylcholine from acetyl-coenzyme A and choline (7). The enzyme is found at pre-synaptic nerve terminals and is a phenotypic marker of cholinergic neurons that are involved in a wide variety of brain functions, such as learning, memory, and sleep (3). Choline acetyltransferase is needed for cholinergic neurons to communicate with target cells and loss of its expression is associated with disorders such as Alzheimer's disease and schizophrenia (7). Additionally, single nucleotide polymorphisms can cause point mutations in choline acetyltransferase that disrupt its kinetic functions, leading to muscle weakness and apnea (2).
Choline acetyltransferase is a monomer (3) consisting of a single subunit that spans 615 residues. According to PDB, its secondary structure is made up of 24 alpha helices and 22 beta strands, which represent 58% of the secondary structure, and the rest of the structure is composed of random coils. The enzyme is composed of two domains, in which each domain is made up of a beta sheet surrounded by alpha helices, and has 2-fold symmetry similar to carnitine acetyltransferase (6).
According to PDB, choline acetyltransferase shares 44% sequence similarity with human carnitine acetyltransferase (PDB ID: 1NM8) with a Z score of 51.6 (5). Since the sequences are more than 30% similar, this means the two proteins also sharesimilar structure. Carnitine acetyltransferase is a family of enzymes that are involved in cellular metabolism through fatty acyl chain transfer. The enzyme acts by facilitating the transfer of an acyl group to carnitine, similar to how choline acetyltransferase catalyzes the transfer of an acetyl group (8). Within the carnitine acetyltransferase family, peroximal carnitine octanoyltransferase (PDB ID: 1XMC.A) has 47% 3D similarity and carnitine O-palmitoyltransferase II (PDB ID: 2DEB.B) has 43% similarity to choline O-acetyltransferase (PDB ID: 1T1U.A). Choline O-acetyltransferase had to be used instead of choline acetyltransferase because there were no explicit alignment results found for 2FY3, but the proteins share 70% sequence similarity.
The two domains of choline acetyltransferase are separated into a binding and catalytic domain. The binding domain consists of residues 1 to 89 and 392 to 615, while the catalytic domain consists of residues 90 to 391 (7). The two domains form a tunnel through the center of choline acetyltransferase, and an important catalytic residue H-324 (1) lies between the two domains within the tunnel. This histidine residue is believed to be a general base catalyst in the reaction transferring the acetyl group of acetyl-CoA to choline, in order to produce the essential neurotransmitter acetylcholine. Acetylcholine is found throughout the central and peripheral nervous systems and is necessary at neuromuscular junctions for muscle movement (3).
One of the ligands of choline acetyltransferase is choline, which attaches to the binding domain through its trimethylammonium group. This trimethylammonium group forms a cation-pi bond with the side chain of Y-552 and also forms van der Waals interactions with the side chains of residues M-84 and Y-436. These interactions are needed in order to position the hydroxyl group of choline to form a hydrogen bond with the imidazole ring of the catalytic residue H-324. While the area surrounding the trimethylammonium group is not relatively tight, the alpha and beta carbons of choline are very tightly surrounded by the walls of the binding domain and the side chains of M-84 and Y-85. This difference in surrounding area explains why there can be modification to the substituents attached to nitrogen without a substantial increase in Km, but variation at the alpha and beta carbons results in such a poor substrate that the Km is very large or undetermined (7).
The other ligands of choline acetyltransferase are acetyl-CoA and CoA, which also interact at the binding domain along with choline. Acetyl-CoA is the lead substrate in the reaction, and the dissociation of CoA is the rate-determining step when ionic strength is low. The negatively charged 3'-phosphate of CoA forms bonds with the positively charged side chains of K-403 and K-407, in which a hydrogen bond is formed with K-407. The pantotheine group of CoA is positioned between two beta-strands, and this separation between the strands exposes amides of residues S-440 and Q-541, which can form two hydrogen bonds with pantotheine amides of CoA. Two more hydrogen bonds are made between the pyrophosphate oxygens of CoA and residues S-412. In all, five hydrogen bonds are formed between the active site and CoA. The acetyl group of acetyl-CoA then forms a bond between its carbonyl oxygen and the side chain of S-540. When both choline and acetyl-CoA are bound at the binding domain, the carbonyl carbon of acetyl-CoA is close enough to undergo nucleophilic attack by the oxygen of choline, which is bonded to H-324. This results in transfer of the acetyl group of acetyl-CoA to choline, which produces acetylcholine (7).
Conformational changes occur in choline acetyltransferase upon binding CoA, which is due to changes in mobility of the P-loop that assists in binding. The P-loop, which is composed of residues 139 to 148, is part of the catalytic domain and reaches around the active site in order to interact with CoA or acetyl-CoA. The interaction involves P-loop residue Q-144 forming two hydrogen bonds with the pyrophosphate oxygens of acetyl-CoA/CoA. In addition, the P-loop forms bonds with the side chains of residues R-442 and R-443. These interactions result in a 1.5 degree rotation of the catalytic domain relative to the binding domain so that the P loop goes from a mobile to anchored state (7). Interestingly, the P-loop is a unique characteristic of choline acetyltransferase and is not present in carnitine acetyltransferase, which has similar structure and function (6). It is possible that the P-loop contributes to the low Km for CoA relative to carnitine acetyltransferase, and it may help in binding of acetyl-CoA when cytosolic levels of the substrate are low (7).
Choline acetyltransferase can also be regulated through phosphorylation of residues located near the binding of the P-loop. A major target of phosphorylation by protein kinase C is S-440, in which phosphorylation of S-440 results in changes in the catalytic activity of choline acetyltransferase (2). Since S-440 is located at the binding site of CoA/acetyl-CoA, the addition of a phosphate group could disrupt binding of the substrate and could also affect binding of the P-loop to the active site (7). Nearby, residue T-456 is also a site of phosphorylation that is targeted by CaM kinase II (2).
Choline acetyltransferase activity can also be affected through modification by drugs such as sulfhydryl-reactive agents. The binding domain contains several cysteine residues that can form disulfide bonds, such as between C-563 and C-550/C-574 and between C-550 and C-574. In addition to C-322, these residues are most likely the sites of modification by the sulfhydryl-reactive agents, which cause decreased activity or complete inactivation of choline acetyltransferase (4). Other drugs that affect the activity of the enzyme are arginine-modifying reagents such as butanedione, which can act on residues R-442 and R-443 that are involved in the interaction of the P-loop in helping to bind CoA or acetyl-CoA (9).