Treatment of prostaglandin endoperoxide (PGH) synthase apoprotein with a 100- or 1000-fold excess of N-acetylimidazole (NAI) led to time-dependent inactivation of both cyclooxygenase and peroxide activities. Reconstitution of apoprotein with heme prior to incubation with NAI substantially protected the enzyme from inactivation. Pretreatment of the protein with either acetylsalicylic acid (aspirin) or (+/-)-2-fluoro-alpha-methyl-4-biphenylacetic acid (flurbiprofen), which inhibit cyclooxygenase activity, did not alter the time course of peroxidase inactivation by NAI. Treatment of NAI-inactivated apoPGH synthase with hydroxylamine led to substantial regeneration of both cyclooxygenase and peroxidase activities. Quantitation of radioactivity following incubation of PGH synthase with [3H-acetyl]NAI indicated incorporation of 1.7 +/- 0.9 acetyl groups/70-kDa subunit. Cleavage of acetylated protein with trypsin under nondenaturing conditions followed by high-performance liquid chromatography analysis demonstrated that most of the radioactivity was incorporated into the 33-kDa fragment although significant radioactivity was also detectable in the 38-kDa fragment. Chymotryptic peptide mapping of acetylated protein revealed numerous potential sites of acetylation distributed in widely divergent regions of the protein. No apparent differences were observed between the chymotryptic maps of apo- and holoenzyme, suggesting that the adduct responsible for loss of catalytic activity is unstable to the chromatographic conditions. The different biochemical properties of PGH synthase acetylated by NAI or aspirin suggest that a major determinant of the specificity of aspirin for Ser530 is binding of the salicylate moiety to this region of the PGH synthase protein.
Derivatives of the potent antiinflammatory agent and cyclooxygenase inhibitor indomethacin were synthesized in which the carboxylic acid moiety was converted into reactive acylating agents. Indomethacin imidazole (indomethacin-IM) and indomethacin N-hydroxysuccinimide (indomethacin-NHS) inactivated both the cyclooxygenase and peroxidase activities when incubated with the apo form of purified prostaglandin endoperoxide synthase (PGH synthase) at a stoichiometry of 1:1. Treatment of the inactivated enzyme with hydroxylamine at neutral pH led to recovery of all peroxidase and about 50% of the cyclooxygenase activity. Hydroxylamine did not regenerate the cyclooxygenase activity of indomethacin-inactivated protein. Reconstitution of the apoprotein with heme protected against inactivation by indomethacin-NHS. Visible spectroscopy established that indomethacin-NHS-inactivated apoenzyme had a reduced capacity to bind heme. Indomethacin-NHS also substantially protected the apoenzyme from cleavage at the trypsin-sensitive Arg277 site. Incubation of [2-14C]indomethacin-NHS with PGH synthase led to incorporation of radioactivity into the protein, but no adduct was detected by reversed-phase HPLC, suggesting it was unstable to the chromatographic conditions. Incubation of indomethacin-NHS with apoprotein followed by HPLC analysis led to the formation of greater amounts of the hydrolysis product indomethacin than did similar treatment of holoprotein. The results suggest that indomethacin-IM and indomethacin-NHS covalently and selectively label PGH synthase near the heme binding site, leading to loss of both catalytic activities of the enzyme.
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