Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin.
Aspirin (acetylsalicylate) treatment of human (h) prostaglandin endoperoxide H synthase (PGHS)-1 expressed in cos-1 cells caused a time-dependent inactivation of oxygenase activity. Aspirin treatment of hPGHS-2 produced an enzyme which retained oxygenase activity but formed exclusively 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) instead of PGH2. The 15-HETE was exclusively of the 15R configuration. The Km values for arachidonate of native and aspirin-treated hPGHS-2 were about the same suggesting that arachidonate binds to both aspirin-treated and native hPGHS-2 in a similar manner. If, as expected, the formation of 15R-HETE proceeds through abstraction of the 13proS hydrogen from arachidonate, O2 insertion must occur from the same side as the hydrogen abstraction; with all other lipoxygenases and cyclooxygenases, O2 addition is antarafacial. When microsomal hPGHS-2 was incubated with [acetyl-14C]aspirin, the enzyme was acetylated. An S516A mutant of hPGHS-2, which retains enzyme activity, was not acetylated. This indicates that Ser-516 is the site of aspirin acetylation of hPGHS-2; this residue is homologous to the "active site" serine of PGHS-1. An S516N mutant of hPGHS-2 was catalytically active; in contrast, an S516Q mutant 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 that this mutant made 15R-HETE as its major product; however, unlike the aspirin-acetylated hPGHS-2, the Km value of the S516M mutant for arachidonate was 100 times that of native hPGHS-2.
Lecomte
M
M
Department of Biochemistry, Michigan State University, East Lansing 48824.
Laneuville
O
O
Ji
C
C
DeWitt
D L
DL
Smith
W L
WL
D000107:Acetylation; D001241:Aspirin / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D016861:Cyclooxygenase Inhibitors / Q000378:metabolism*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides
D016861:Cyclooxygenase Inhibitors; D006893:Hydroxyeicosatetraenoic Acids; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D001241:Aspirin