Cyclooxygenase Structure and Mechanism

How Aspirin and NSAIDs Work

Prostaglandins and NSAIDS
Prostaglandins are potent mediators of inflammation. The first and committed step in the production of prostaglandins from arachidonic acid is the bis-oxygenation of arachindonate to prostaglandin PGG2. This is followed by reduction to PGH2 in a peroxidase reaction. Both these reactions are catalyzed by cyclooxygenase, also known as PGH synthase.

Cyclooxygenase (COX) is inhibited by the family of drugs known as non-steroidal anti-inflammatory drugs or NSAIDs. Aspirin, ibuprofen, flurbiprofen and acetaminophen (trade name Tylenol) are all NSAIDs.

There are two isoforms of COX in animals: COX-1, which carries out normal, physiological production of prostaglandins, and COX-2, which is induced by cytokines, mitogens and endotoxins in inflammatory cells, and which is responsible for the production of prostaglandins in inflammation.

The structure shown at left is that of COX-1 from sheep, inactivated by bromoaspirin, the structure of which is shown below.

The Enzyme Structure

The first 24 residues of COX-1 are a signal sequence. This domain is removed in the mature enzyme and will not be discussed here. Similarly , residues 25-32 do not yield interpretable electron density, and are not shown in the structure shown.

The remaining 551 residues of the enzyme (residues 33-583) comprise three distinct domains. The first of these, residues 33-72, form a small compact module that is similar to epidermal growth factor .

The second domain, composed of residues 73-116, forms a right-handed spiral of four alpha-helical segments along one side of the protein . . This domain of the protein forms a membrane-binding motif. The helical segments are amphipathic, with most of the hydrophobic residues (shown in green ) facing away from the protein, where they can interact with a lipid bilayer.

Turn off the hydrophobic residues and we will consider the third domain of the COX enzyme, the catalytic domain (in blue), a globular structure that contains both the cyclooxygenase and peroxidase active sites .

The peroxidase site includes a heme .

The iron(III) in the center of this heme is coordinated by His-388
and by His-207 .

Let's return to our view of the whole molecule .

The cyclooxygenase active site lies at the end of a long, narrow, hydrophobic tunnel or channel. Three of the alpha helices of the membrane-binding domain lie at the entrance to this tunnel. .

In this bromoaspirin-inactivated structure, Ser-530 is bromoacetylated , and a molecule of salicylate is bound in the tunnel

Deep in the tunnel, at the far end, lies Tyr-385, a catalytically important residue. Heme-dependent peroxidase activity is implicated in the formation of a proposed Tyr-385 radical, which is required for cyclooxygenase activity.

Now take another look at the tunnel with the bromoacetyl-Ser 530, the salicylate, and the essential Tyr-385 all shown within the tunnel.

At this point in this exercise, you are literally looking at the view an arachidonic acid substrate has of the active site at the end of the tunnel. The yellow helices, you will recall form the membrane interface. Arachidonic acid substrates flow up into the tunnel from the membrane interior.

With this view, it should also be clear why aspirin and other NSAIDs block the synthesis of prostaglandins. In various ways, they all act by filling and blocking the tunnel, preventing the migration of arachidonic acid to the active site at the back of the tunnel.

There are thought to be at least four different mechanisms of action for NSAIDs. Aspirin (and also bromoaspirin) is the only one which covalently modifies a residue in the tunnel, thus irreversibly inactivating both COX-1 and COX-2.

Ibuprofen (shown below) acts instead by competing in a reversible fashion for the substrate binding site in the tunnel.

Flurbiprofen and indomethacin, members of the third class of inhibitors, are shown below.

Fluribiprofen and indomethacin cause a slow, time-dependent inhibition of COX-1 and COX-2, apparently via formation of a salt bridge between a carboxylate on the drug and Arg-120(shown here in green), which lies in the tunnel.

The drug SC-558 acts by a fourth mechanism, specifically inhibiting COX-2. It is a weak competitive inhibitor of COX-1 but inhibits COX-2 in a slow, time-dependent process. Specific COX-2 inhibitors will likely be the drugs of the future, since they will be able to selectively block the inflammation mediated by COX-2, without the potential for stomach lesions and renal toxicity that arise from COX-1 inhibition.