PGH synthase

In order for the cyclooxygenase to function, a source of hydroperoxide (R—O—O—H) appears to be required. The hydroperoxide oxidizes a heme prosthetic group at the peroxidase active site of PGH synthase. This in turn leads to the oxidation of a tyrosine residue producing a tyrosine radical which is apparendy involved in the abstraction of the 13-pro-(5)-hydrogen of AA (25). The cyclooxygenase is inactivated during catalysis by the nonproductive breakdown of an active enzyme intermediate. This suicide inactivation occurs, on average, every 1400 catalytic turnovers. 

Fig. 3. Putative mechanism of PGH synthase action on arachidonic acid.

Incubation of BP with arachidonic acid and ram seminal vesicle micro-somes, a rich source of PGH synthase, produces 1,6-, 3,6-, and 6,12-quinones as the exclusive products of oxidation (Figure 2) (18). 

These are the same quinones that are formed when 6-hydroxy-BP is oxidized by air or microsomes (19). However, there is no definitive evidence that 6-hydroxy-BP is an intermediate in their formation by PGH synthase. Among all of the stable metabolites of BP, the quinones are distinctive because, unlike phenols and dihydrodiols, they are not derived from arene oxides. Thus, arene oxides do not appear to be products of BP oxidation by PGH synthase (19,20). 

Potent inhibition of PGH synthase-dependent BP oxidation by antioxidants suggests that the quinones are products of free radical reactions (18 ). ... 

Addition of RNA or DNA prior to oxidation of BP by PGH synthase results in substantial nucleic acid binding (17,21). Addition of RNA five minutes after initiation of oxidation leads to no covalent binding (17). This implies that the quinones do not bind to nucleic acid but rather a short-lived intermediate in their formation does. Arachidonic acid oxygenation in ram seminal vesicle microsomes is complete within two min, which suggests that the reactive intermediate is generated concurrently with PGH2 The structures of the nucleic acid adducts have not been elucidated so the identity of the reactive intermediate is unknown. 

When the 7,8-hydroxyl groups are missing, epoxide introduction occurs from both sides of the pyrene ring. Thus 7,8-dihydrobenzo(a)-pyrene is cooxidized by PGH synthase to a potent mutagen that is identified by product and nucleic acid binding studies as 9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (Equation 3) (32). The structures... 

These findings indicate that PGH synthase in the presence of arachidonate can catalyze the terminal activation step in BP carcinogenesis and that the reaction may be general for dihydrodiol metabolites of polycyclic hydrocarbons. Guthrie et. al. have shown that PGH synthase catalyzes the activation of chrysene and benzanthracene dihydrodiols to potent mutagens (33). As in the case with BP, only the dihydrodiol that is a precursor to bay region diol epoxides is activated. We have recently shown that 3,4-dihydroxy-3,4-dihydro-benzo(a)anthracene is oxidized by PGH synthase to tetrahydrotetraols derived from the anti-diol epoxide (Equation 4) (34). 

PGH synthase and the related enzyme lipoxygenase occupy a position at the interface of peroxidase chemistry and free radical chemistry and can clearly trigger metabolic activation by both mechanisms. The peroxidase pathway activates compounds such as diethylstilbestrol and aromatic amines whereas the free radical pathway activates polycyclic hydrocarbons (59). Both pathways require synthesis of hydroperoxide in order to trigger oxidation. 

Cyciooxygenase (COX) PGH synthase the first enzyme in the generation of the eicosanoids from polyunsaturated fatty acids. 

These observations suggest that vitamin E may play a regulatory role in PG biosynthesis by controlling the formation of key intermediates such as hydroperoxides and cyclic endoperoxides. In these experiments, peroxidase activity associated with the PGH synthase could not be measured because of the contamination of other peroxidases like cyt P-450 hydroperoxidase in crude microsomal preparations. Our attempts to measure the differences in catalytic rates employing indomethacin were not successful. Nevertheless, it is not too unreasonable to assume that probably both activities of PGH synthase are equally affected by vitamin E deficiency, since both require heme as cofactor. 

Geminal difluoro-substituted analogues of arachidonic acid have been used as substrates for PGH synthase as an alternative approach to difluoro-analogues of TXA2 and PGI2, with unexpected but useful outcomes. For example, although 10,10-difluo-... 

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