ACETYLATION OF HUMAN PROSTAGLANDIN ENDOPEROXIDE SYNTHASE-2 (CYCLOOXYGENASE-2) BY ASPIRIN

Aspirin (acetylsalicylate) treatment of human (h) prostaglandin endope roxide H synthase (PGHS)-1 expressed in cos-1 cells caused a time-depe ndent inactivation of oxygenase activity. Aspirin treatment of hPGHS-2 produced an enzyme which retained oxygenase activity but formed exclu sively 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15 HETE) instead of PGH(2). The 15-HETE was exclusively of the 15R configuration. The K-m values for arachidonate of native and aspirin-treated hPGHS-2 were ab out the same suggesting that arachidonate binds to both aspirin-treate d and native hPGHS-2 in a similar manner. If, as expected, the formati on of 15R-HETE proceeds through abstraction of the 13proS hydrogen fro m arachidonate, O-2, insertion must occur from the same side as the hy drogen abstraction; with all other lipoxy-genases and cyclooxygenases, O-2 addition is antarafacial. When microsomal hPGHS-2 was incubated w ith [acetyl-C-14]aspirin, the enzyme was acetylated. An S516A mutant o f hPGHS-2, which retains enzyme activity, was not acetylated. This ind icates that Ser-516 is the site of aspirin acetylation of PGHS-2; this residue is homologous to the ''active site'' serine of PGHS-1. An S51 6N mutant of hPGHS-2 was catalytically active; in contrast, an S516Q m utant lacked cyclooxygenase but retained peroxidase activity. Because in the case of PGHS-1 a smaller asparagine substitution is sufficient to eliminate cyclooxygenase activity, we conclude that the active site of PGHS-2 is slightly larger than that of PGHS-1. An S516M mutant of hPGHS-2 was obtained which resembled aspirin-acetylated hPGHS-2 in tha t this mutant made 15R-HETE as its major product; however, unlike the aspirin-acetylated hPGHS-2, the K-m value of the S516M mutant for arac hidonate was 100 times that of native hPGHS-2.