Acetylation of prostaglandin endoperoxide synthase by N-acetylimidazole: comparison to acetylation by aspirin.

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.