Mutation of serine-516 in human prostaglandin G/H synthase-2 to methionine or aspirin acetylation of this residue stimulates 15- R-HETE synthesis

Prostaglandin G/H synthase (PGHS) is a key enzyme in cellular prostaglandin (PG) synthesis and is the target of non-steroidal anti-inflammatory agents. PGHS occurs in two isoforms, termed PGHS-1 and PGHS-2. These isoforms differ in several respects, including their enzymatic activity following acetylation by aspirin. While PG synthesis by both isoforms is inhibited by aspirin, 15- R-hydroxyeicosatetraenoic acid (15- R-HETE) synthesis by PGHS-2, but not PGHS-1, is stimulated by preincubation with aspirin. We have mutated the putative aspirin acetylation site of hPGHS-2, and expressed the mutants in COS-7 cells using recombinant vaccinia virus. Enzyme activity and inhibitor sensitivity studies provide evidence that Ser 516 is the aspirin acetylation site of human PGHS-2 and that substitution of a methionine residue at this position can mimic the effects of aspirin acetylation on enzyme activity.