Introduction
Figure 1.Tetrameric dimer of dimer for ACAT
Acyl-coenzyme A: cholesterol acyltransferase (ACAT), also known as Human Sterol O-acyltransferase (hSOAT) is an enzyme that catalyzes the reaction between long chain
fatty acyl CoA and intracellular
cholesterol to form the more hydrophobic cholesteryl ester for cholesterol storage.
Cholesteryl ester is the primary form of how cholesterol is stored in multiple types of cells and transported through the circulatory system. ACAT is an endoplasmic reticulum membrane protein, and ACAT is a part of the
MBOAT (membrane-bound O-acyltransferase) family, which also includes acyl-coenzyme A: diacylglycerol acyltransferase (
DGAT) and ghrelin O-acyltransferase (
GOAT).
There have been two ACAT isoforms discovered in mammals, ACAT1 and ACAT2, and they are predominantly located in different parts of the body. ACAT1 is mainly found in the liver, kidneys, adrenal glands and macrophages, whereas ACAT2 is found only in the intestines and liver.
Structure
Overall Structure
ACAT is a tetramer composed of a dimer of a dimer, but is able to perform its function solely as a . There are in each domain which create a tunnel for the active site. There are also three helices found on the intracellular side (IH1, IH2, and IH3) and one helix on the extracellular side (EH1). The active site contains three tunnels – the transmembrane tunnel for cholesterol entrance, the cytosolic tunnel for acyl-CoA entrance, and the lumen tunnel for cholesterol ester exit. ACAT also has an amino-terminal cytosolic domain (NTD) that is important for tetramerization of this protein.
Important Residues
An important residue in the ACAT active site is His460, a Histidine, which is located in the middle of the tunnels. It is thought that His460 is located on TM7 (Qian et al.). When converting to a cholesteryl ester, the acts as a catalytic base that deprotonates the cholesterol. An asparagine Asn421 is another important residue in the reaction that is able to form a hydrogen bond with acyl-CoA for stabilization.
Important Interactions
Between the two protomers in each dimer, Van der Waals interactions occur between TM1 of one protomer and the lumenal TM6 and the cytosolic TM9 of the other protomer. The two dimers make limited contact within the membrane through an interface that is thought to be located between TM2, TM5, TM6 and IH2.
Proposed Mechanism
Due to limited high-resolution structural representations of ACAT, its mechanism remains ambiguous. However, the general mechanism involving the substrates and products of ACAT is understood
[1].
Figure 2: Proposed mechanism for ACAT by Guan et al.
In this reaction,
oleoyl-CoA and cholesterol are the reactants and they undergo the reaction catalyzed by ACAT to form cholesteryl-oleate which is used as a storage form of cholesterol. The hydroxyl group on cholesterol is deprotonated, then attacks the
thioester bond of oleoyl-CoA, kicking off CoA-SH as a leaving group. This mechanism is shown in Figure 2.
Figure 3: Mechanism for ACAT proposed by Qian et al.
However, Qian et al.[2] proposed a mechanism involving the important residues His460 and Asn421. In this mechanism, His460 acts as a general base to deprotonate the hydroxyl group on cholesterol, activating it as a nucleophile. Then, Asn421 possibly forms a hydrogen bond with oleoyl-CoA to stabilize the reaction. This mechanism is shown in Figure 3.
Active Site
Allosteric Binding Pocket
ACATs are enzymes that can be allosterically activated by sterol molecules like cholesterol. There are two proposed binding sites for cholesterol, with each binding site being distinctively different. One site is the substrate active site mentioned above and the other site is the allosteric binding site. The allosteric binding site has the ability to direct feedback regulation over the concentration of cholesterol in the endoplasmic reticulum.
In order to act as an allosteric activator, the sterol molecules must contain a highly conserved 3-beta hydroxyl group at the first steroid ring (ring A) [3] . An activator must also contain a conserved iso-octyl side chain to efficiently activate ACAT. The conserved 3-beta hydroxyl group allows for binding of the cholesterol to cause conformational changes to the ACAT dimer. Upon conformational changes, the rate of esterification is increased.
Tunnels
The nine transmembrane segments create a cytosolic tunnel and a transmembrane tunnel that meet at the site of catalysis (active site). Acyl-coenzyme A enters the active site though the cytosolic tunnel and cholesterol enters through the transmembrane tunnel. These then meet in the catalytic site to react and form the cholesteryl ester. When exiting the catalytic site, the free CoA is able to release through the cytosolic tunnel to the cytosol and the cholesterol ester is able to release through the to the membrane or through the lumen tunnel to the lumen. Certain residues that line the transmembrane tunnel are important in ACAT activity (E259, E263, R262, P304, L306, V423, V424, M265, I261, and H460).
Medical Relevance
The mechanism of ACAT is essential for cholesterol storage and cholesterol transfer through the plasma because cholesteryl ester is the primary form of cholesterol used for these events. Additionally, ACAT can use a variety of different sterol molecules besides cholesterol as substrates and activators. Because of its biological importance, ACAT has been linked to atherosclerosis, Alzheimer’s disease, and cancer as a potential drug target for treatment of these diseases. Various studies have looked into ACAT inhibition and how that inhibition treats or prevents certain diseases, such as reducing the size and metastasis of certain tumors and reducing the formation of plaques in atherosclerosis. ACAT is an important target for these diseases due to its functional relevance in cholesterol metabolism.
