Cyclooxygenase
Created by Amanda Mullen
Cyclooxygenase-2(1CX2) from Mus musculus is an enzyme that catalyzes the oxidation of arachidonic acid to ProstaglandinG2, followed by the reduction of this product to form ProstaglandinH2 (PGH2) (1). This reaction is the first committed step in the production of thromboxane, prostacyclin, and all prostaglandins. These compounds are important biolipids with gastrointestinal, renal, and cardiovascular physiological functions. (2). PGH2 specifically acts as a mediator of inflammation; thus, the inhibition of this reaction decreases the inflammatory response. For this reason, many non-steroidal anti-inflammatory drugs target cyclooxygenase-2 (1). The molecular weight of COX-2 is 268,905.75 g/mol, and its isoelectric point (pI) is 6.88.
The structure of cyclooxygenase-2 directly correlates with the function of the enzyme. COX-2 exists physiologically as a tetramer with four identical subunits (1). Each subunit is broken down into three distinct domains: epidermal growth factor, membrane, and catalytic. The epidermal growth factor domain covers residues 33-72 and is involved in signal transduction. The membrane domain extends from residue 73-116 and is responsible for membrane interaction via amphipathic helical segments. These helices contain both hydrophobic and polar residues, but the hydrophobic residues (shown in yellow) face away from the protein so they can interact with the lipid bilayers of membranes. The membrane domain makes a hydrophobic tunnel leading to the active sites in the catalytic domain. The catalytic domain is composed of residues 117-583 and contains both the cyclooxygenase and peroxidase active sites and the two ligand components (6).
Cyclooxygenase-2 has two functions: the oxidation of arachidonic acid and the reduction of prostaglandinG2 (PGG2) to prostaglandinH2 (PGH2). To complete both reactions (see Reaction_Scheme), there are two separate but functionally linked active sites within the catalytic domain of COX-2. The cyclooxygenase active site converts arachidonic acid to PGG2 (5). This active site is located at the end of a long hydrophobic channel (orange) that extends away from the membrane domain toward the heme group (4). The carboxylate end of arachidonic acid forms hydrogen bonds with the side chains of Tyr-385 and Ser-530, and the w-end of arachidonic acid participates in van der Waals interactions with the side chain of Arg-120. As arachidonic acid moves down the hydrophobic channel, Tyr-385 forms a tyrosine radical that oxidizes the carbon-13 of arachidonic acid forming PGG2(3). The prostaglandinG2 migrates to the peroxidase active site where it undergoes a two electron reduction. PGG2 binds to His-207 and Gln-203; as a result, the peroxide bond is broken. The iron in the heme ligand component reduces the peroxide in PGG2 to an alcohol making PGH2(7).
Since arachidonic acid metabolism is a precursor for inflammation, many non-steroidal anti-inflammatory drugs (NSAIDs) target the cyclooxygenase active site of COX-2. There are four distinct mechanisms by which NSAIDs inhibit this reaction: irreversible inactivation, reversible competitive inhibition, ionic interaction, and time-dependant COX-2 selective inhibition. Aspirin (pdb ID = 3N8Y) acetylates Ser-530 on the cyclooxygenase active site; acetylation irreversibly inactivates the enzyme because the covalent change interferes with arachidonic acid binding (1). The figure shows Ser-530 (green) superimposed on the acetylated version of the serine (red) after aspirin (2-hydroxybenzoic acid) binding. An example of reversible competitive inhibition is naproxen (pdb ID = 3NT1) which forms hydrogen bonds with Arg-120 and Tyr-355 in the COX active site. Naproxen competes with arachidonic acid for the COX active site (8). Drugs, such as indomethacin (pdb ID=4COX), block the active site by forming a salt bridge between the positively-charged side chain of Arg-120 and the carboxylate of the drug. SC-558 (pdb ID = 1cx2) is a COX-2 selective inhibitor that binds to a specific pocket in the hydrophobic channel. The bromophenyl ring of SC-558 binds to Leu-352, Tyr-355, Phe-518 and Val-523 (1). The other three drug complexes are not specific to COX-2; they also inhibit the COX site in COX-1. SC-558 cannot inhibit COX-1 because Ile-523 blocks the binding site.
The two ligand components for COX-2 are protoporphyrin IX containing Fe (HEME) and 1-phenylsulfonamide-3-trifluoromethyl-5-parabromophenylpyrazole (S-58 selective inhibitor). Heme, a prosthetic group, is involved in both the peroxidase and cyclooxygenase reactions. In the cyclooxygenase active site, the heme binds to His-388 and interacts with Tyr-385 forming a tyrosine radical which is necessary for the formation of PGG2 from arachidonic acid. In the peroxidase active site, heme acts as the reducing agent in the reduction of PGG2 to form PGH2 (2). S-58 or SC-558 is a selective inhibitor complexed with cyclooxygenase-2 in this crystallization (1).
The secondary structure of COX-2 contains 23 alpha-helices (red) and two anti parallel beta-sheets (yellow). The three domains have specific three-dimensional structures that determine their individual function. The EGF domain contains two anti-parallel beta-sheets and is located at the tetramer interface. The membrane domain is composed of four alpha-helices (A-D) containing hydrophobic residues projected away from the protein toward the membrane. Helix-D extends into the catalytic domain forming a hydrophobic tunnel leading to the COX active site. The catalytic domain contains a helix bundle (shown in red) that facilitates the heme-binding site, peroxidase active site, and cyclooxygenase active site (7).
Cyclooxygenase-1 (pdb ID= 1EQG), shown in purple, has an approximate 60% sequence similarity to cylcooxygenase-2 (blue) and an almost identical folding pattern. Results from the DALI Server (Z=57.5, rmsd= 1.0) and protein Basic Local Alignment Search Tool (BlastP) (E=0.001) searches show that COX-1 has both primary and tertiary similarities to COX-2. The cyclooxygenase and peroxidase active sites show 87% sequence conservation in both enzymes. The structural differences between COX-1 and COX-2 are subtle with little effect on function, but the changes effect specific inhibition. At the junction between helices C and D of the membrane domain, COX-1 contains a threonine instead of a proline at position 106. This proline insertion only causes a small change in the local structure. The hydrophobic channel in COX-1 at position 523 has an isoleucine; whereas, COX-2 has a valine. The Ile-523 in COX-1 prevents inhibitors from binding allowing for COX-2 specific inhibition by inhibitors such as SC-558(1).