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

Abstract

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.

Rapid Inactivation of Prostaglandin Endoperoxide Synthases by N-(Carboxyalkyl)maleimides

“…Thus, A-(carboxyheptyl)maleimide is between 5 and 3 orders of magnitude more potent than aspirin, depending on the time of preincubation. The exquisite sensitivity of cyclooxygenase inhibition to the structure of the substituted maleimides is consistent with our previous suggestion that the sensitivity of Ser530 to acetylation by aspirin is dependent on complementary interactions between the salicylate moiety and PGHS (Wells & Marnett, 1992). Furthermore, it raises the possibility that other covalent inactivators could be designed to differentially inhibit the two isoforms of PGHS.…”

“…Covalent modification of PGHS is responsible for enzyme inactivation by acetylsalicylic acid (Robinson & Vane, 1974;Rome et al, 1976), acylimidazoles (Wells & Marnett, 1992Scherer et al, 1992), acyl-A-hydroxysuccinimides (Wells & Marnett, 1993), and A-alkylmaleimides (Kennedy et al, 1993). In all these cases, inactivation is time-dependent and occurs at ratios of inhibitor/enzyme of 100 or greater.…”

The mode of action of aspirin-like drugs: effect on inducible nitric oxide synthase.

“…These experiments further indicate that acetylation of iNOS [and/or essential cofactor(s)] inactivates its catalytic activity and that the potency of NAI is relatively greater than that of aspirin. NAI, which is commonly used for acetylation of tyrosine hydroxyl (28)(29)(30), acetylates protein residues at rates proportional to their nucleophilicity and accessibility (27,28).…”

Theory and Applications of Covalent Docking in Drug Discovery: Merits and Pitfalls

“…It is reported that approximately, 33% of the covalent drugs in the market are anti-infectives (most notably the β-lactam class of antibiotics), 20% treat cancer, 15% treat gastrointestinal disorders, and ~15% are used to treat central nervous system and cardiovascular indications [ 14 ]. The earliest example of a covalent drug is aspirin, which was first marketed over a century ago; aspirin covalently modifies cyclooxygenase by inducing the acetylation of a serine residue that is situated in the active site ( Figure 1 A) [ 15 , 16 , 17 , 18 ]. β-lactam antibiotics are other examples of a covalent drug which acylate the active site serine of penicillin-binding proteins (PBPs) and kill the bacteria by inhibiting the final step of cell wall biosynthesis.…”