The cyclooxygenases

Abstract

Cyclooxygenases (COXs) catalyze the rate-limiting step in the production of prostaglandins, bioactive compounds involved in processes such as fever and sensitivity to pain, and are the target of aspirin-like drugs. COX genes have been cloned from coral, tunicates and vertebrates, and in all the phyla where they are found, there are two genes encoding two COX isoenzymes; it is unclear whether these genes arose from an early single duplication event or from multiple independent duplications in evolution. The intron-exon arrangement of COX genes is completely conserved in vertebrates and mostly conserved in all species. Exon boundaries largely define the four functional domains of the encoded protein: the amino-terminal hydrophobic signal peptide, the dimerization domain, the membrane-binding domain, and the catalytic domain. The catalytic domain of each enzyme contains distinct peroxidase and cyclooxygenase active sites; COXs are classified as members of the myeloperoxidase family. All COXs are homodimers and monotopic membrane proteins (inserted into only one leaflet of the membrane), and they appear to be targeted to the lumenal membrane of the endoplasmic reticulum, where they are N-glycosylated. In mammals, the two COX genes encode a constitutive isoenzyme (COX-1) and an inducible isoenzyme (COX-2); both are of significant pharmacological importance.

Figure 1

Primary structures of COX genes and COX proteins. (a) Schematic representation of human COX-1 and COX-2 genes and the mRNAs they encode (shown as white bars below the genes). Black boxes in the genes and white boxes in the mRNAs denote exons; numbers above each gene are exon numbers while numbers within the white boxes indicate the size of each exon in nucleotides; single lines in the genes indicate introns and untranslated regions of first and last exons (the latter being shown as gray boxes in the mRNAs). Adapted from [10]. (b) Schematic representation of human COX proteins (all known vertebrate proteins have the same general arrangement). Numbers denote amino-acid residues; the exons encoding each domain are shown on bars below the proteins; important residues are indicated as shown in the key (and with letters in the single-letter amino-acid code, with a subscript number indicating the residue number). Sp, signal peptide; Dm, dimerization domain; EG; epidermal growth factor domain; Mb, membrane-binding domain; Cat, catalytic domain.

Figure 2

Cross-section of a cyclooxygenase monomer in the lumen of the endoplasmic reticulum, showing the two distinct catalytic sites. Cx, cyclooxygenase catalytic site; Mb, membrane-binding domain; Px, peroxidase catalytic site.

Figure 3

Production of prostaglandins by COXs. (a) The two reactions performed by cyclooxygenases: the conversion of arachidonic acid to prostaglandin G₂ by the cyclooxygenase activity and the conversion of prostaglandin G₂ to prostaglandin H₂ by the peroxidase activity. (b) The cell-specific synthases that are involved in the conversion of prostaglandin H₂ to the five principal prostaglandins. (c) The reaction mechanism of COX-1. (1) First, a ferryl-oxo (FeIV) protoporphyrin radical in the heme in the peroxidase active site is produced when endogenous oxidant(s) oxidizes ferric heme (FeIII) to ferryl-oxo (FeIV) protoporphyrin radical through a two-electron oxidation. (2) The Tyr384 residue in the cyclooxygenase active site is activated, through a single-electron reduction reaction with the FeIV protoporphyrin radical, to produce a tyrosyl radical. In the first step of the oxygenation process (3), the 13-pro(S) hydrogen of arachidonic acid in the COX site is abstracted by the tyrosyl radical to produce the arachidonyl radical. (4) This is followed by the reaction of the arachidonyl radical with two molecules of oxygen, to yield prostaglandin G₂. (5) Prostaglandin G₂ then diffuses (dotted line) to the peroxidase active site and is reduced to prostaglandin H₂ by the peroxidase activity (1). AA, arachidonic acid; EnR, an endogenous reductant; Fe⁺⁺⁺, ferric heme; Fe=O ⁺⁺⁺⁺, Ferryl-oxo FeIV porphyrin radical; Tyr-OH, active site tyrosine; Tyr-O, tyrosyl radical.