Prostaglandin H synthase. Stoichiometry of heme cofactor.
The stoichiometry of heme interaction with prostaglandin H synthase was determined by titration of the apoenzyme purified from sheep seminal vesicles. Maximal cyclooxygenase activity was reached when 0.53 +/- 0.11 (n = 6) heme/70,000-Da subunit had been added. Spectrophotometric titrations at 411 nm showed a transition when 0.53 +/- 0.04 (n = 5) heme/subunit had been added. The results from the titration end points were corroborated by comparison of the specific cyclooxygenase activity based on subunit concentration with the specific activity/mol of heme (calculated from the incremental increases in activity during the titration). The value based on subunit was approximately half (0.58 +/- 0.11; n = 6) that based on heme, consistent with one heme/two subunits. Analysis of synthase holoenzyme after chromatography on DEAE-cellulose provided validation for the concept that only one subunit needs to bind heme to give a catalytically active synthase dimer. Binding of some heme to the second subunit appears to be only coincidentally associated with complete saturation of the active subunit. Titrations of the synthase with Mn-protoporphyrin IX gave results which confirmed the presence of two high affinity metalloporphyrin sites/dimer. Unlike heme, two Mn-protoporphyrin IX need be bound per dimer to obtain full catalytic activity. Prostaglandin H synthase appears to have two high affinity binding sites for metalloporphyrins. The two sites have slightly different affinities for heme. The synthase dimer is capable of cyclooxygenase catalysis when the site with higher affinity is occupied by heme. The two subunits of the enzyme are thus not completely identical.
D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011485:Protein Binding; D011524:Protoporphyrins / Q000494:pharmacology; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep; D013053:Spectrophotometry
D001051:Apoenzymes; D046911:Macromolecular Substances; D011524:Protoporphyrins; D006418:Heme; C028025:protoporphyrin IX; D011451:Prostaglandin-Endoperoxide Synthases