Cyclo Oxygenase Biochemistry

However, our main concern here is with the cyclo-oxygenase enzyme itself. It had been recognised since the 1960s that tissue homogenates generated prostaglandins and that the enzyme activity seemed to be located in the "high-speed" membrane fraction, but it wasn't until the mid 1970s that convincing data were obtained about the intracellular location of the cyclo-oxygenase. Experiments such as those performed by Bohman and Larsson in 1975 (11) on the rabbit kidney suggested that the enzyme was associated with the endoplasmic reticulum. This membrane-bound location of the cyclo-oxygenase complex frustrated many early efforts to characterise this haem-containing enzyme. The subsequent discovery that it could be solubilized using detergents was important in providing a more convenient source of enzyme that could be subjected to further analysis. Useful data were gathered by Roth et al. (12) who ingeniously adapted Vane's finding (see below) to assess the molecular weight of the enzyme by using radioactive aspirin, labelled with tritium on the labile acetyl group. After treatment, a tissue extract contained a labelled protein with a molecular weight of 85 kDa.

As a result of the "molecular revolution" we have all benefited enormously from the cloning and sequencing of cyclo-oxygenase in the late 1980s (13, 14) and the extraordinary discovery of a further Cox isoforms in 1990 - again as the result of work by several laboratories published almost simultaneously. Although presaged to some extent by pharmacological data, the actual discovery of the new isoform came from researches into the nature of inducible genes. While investigating the expression of early-response genes in fibroblasts transformed with Rous sarcoma virus, Simmons and his colleagues (15) identified a novel mRNA transcript that coded for a protein that was not identical but which had a high sequence similarity to the seminal vesicle Cox enzyme and suggested that a Cox isozyme had been discovered. Likewise, while studying phorbol-ester-induced genes in Swiss 3T3 cells Herschman and colleagues (16) also observed a novel cDNA species encoding a protein with a predicted structure similar to Cox-1. The same laboratory confirmed that this gene product was indeed a novel Cox and that its induction was inhibited by dexamethasone. Similarly findings were subsequently reported in several other cell types.

The two cyclo-oxygenases were accordingly renamed as Cox-1, referring to the original enzyme isolated from seminal vesicles and subsequently found to be distributed almost ubiquitously and Cox-2, denoting the "inducible" form of the enzyme (although it was expressed basally in the brain and elsewhere). The two genes had a different chromosomal organization in rodents and humans, and Cox-l/Cox-2 mRNA was differentially expressed in human tissues (17). Promoter analysis confirmed a fundamental difference between the two isozymes, with the Cox-2 promoter containing elements strongly reminiscent of those genes that are activated during cellular stress and down-regulated by glucocorticoids, whereas the Cox-l promoter had the appearance of a "housekeeping" gene (18). Histological and other studies confirmed this apparent division of labour between the two enzymes, and Cox-1 seemed to be the predominant isoform in healthy gastrointestinal tissue from several species.

Mechanistically, both Cox isoforms are bifunctional enzymes and have two distinct catalytic activities. The first, dioxygenase step incorporates two molecules of oxygen into the arachidonic (or other fatty acid substrate) chain at C-11 and C-15. This allows a cyclisation and the formation of the highly unstable endoperoxide intermediate prostaglandin G2, which bears a hydroperoxy group at C-15. The second, peroxidase, function of the enzyme converts this to prostaglandin H2 having a hydroxygroup at C-15. This moiety can then be transformed in a cell-specific manner by separate isomerase, reductase or synthase enzymes into other prostanoids. Both Cox-1 and Cox-2 probably exist as homodimers attached to intracellular membranes.

The subsequent solution of the crystal structure of any enzyme is itself a landmark achievement, and the case of the cyclo-oxygenases was to shed much light on the mechanism of the selective Cox-2 inhibitors (see below) (19, 20). Despite their high homology, detailed examination of the structure of the catalytic sites revealed the substrate binding "channel" in the two enzymes to be quite different. A single amino-acid change, from the comparatively bulky Ile in Cox-l to Val at position 523 in Cox-2 (equivalent to position 509 in Cox-1), and the conformational changes that this produced resulted in enhanced access to a bulging "side pocket" in Cox-2 that was not present in Cox-1.

More recently still, further potential cyclo-oxygenase variants (21) have been identified. These may prove important in understanding the biology of the enzyme in the future.

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