Inactivation of Prostaglandin Endoperoxide Synthase (PGHS) by N-(Substituted)Maleimides

Abstract

Prostaglandin endoperoxide synthase (PGHS, EC 1.14.99.1) catalyzes the first two steps of prostaglandin biosynthesis.¹ Its cyclooxygenase activity oxygenates arachidonic acid to form PGG₂; the peroxidase activity of the enzyme then reduces PGG₂ in the presence of a reducing substrate to the corresponding alcohol PGH₂.^2,3 The cyclooxygenase activity is inhibited by a structurally diverse class of compounds known as non-steroidal antiinflammatory drugs (NSAID’s).⁴ Inhibition of PGHS by NSAID’s is believed to be the biochemical basis for their antiinflammatory properties.⁵ Covalent modification of PGHS is responsible for enzyme inactivation by aspirin⁴, acylimidazoles⁶, acyl-N-hydroxysuccinimides^6,7, and N-alkylmaleimides.⁸ However, a large excess of inhibitor (~300 fold) and prolonged incubation times (~120 min) are required to achieve significant inhibition in all of these cases. In the present study, we have tethered to the maleimido moiety, a series of substrate and inhibitor mimics that are capable of binding at the fatty acid substrate site. Maleimide derivatives linked to a series of medium length fatty acids were found to exhibit much more potent cyclooxygenase inactivation. The most potent of these inhibitors, N-(carboxyheptyl)maleimide, inhibits enyzme activity within seconds after mixing with a stoichiometric amount of PGHS protein. Varying the length of the alkyl chain in N-(carboxyalkyl)maleimides or removal of the carboxylate dramatically reduces their potency as rapid PGHS inhibitors. In addition to the structure-activity relationships, the mechanism of inhibition of PGHS by N-(carboxyheptyl)maleimide was explored by subsequent inhibition studies with radiolabeled N-(carboxyheptyl)maleimide. Although incubation of apoPGHS with radiolabeled inhibitor led to the incorporation of radioactivity in the protein, subsequent attempts to identify the amino acid residue(s) by peptide mapping were unsuccessful, presumably due to the instability of the enzyme/inhibitor adduct.