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| ACETYLATION OF HUMAN PROSTAGLANDIN ENDOPEROXIDE SYNTHASE-2 (CYCLOOXYGENASE-2) BY ASPIRIN |
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| Author(s): LECOMTE M, LANEUVILLE O, JI C, DEWITT DL, SMITH WL |
| Source: JOURNAL OF BIOLOGICAL CHEMISTRY Volume: 269 Issue: 18 Pages: 13207-13215 Published: MAY 6 1994 |
| Times Cited: 228 References: 57 |
| Abstract: 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 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 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, O-2, insertion must occur from the same side as the hydrogen abstraction; with all other lipoxy-genases and cyclooxygenases, O-2 addition is antarafacial. When microsomal hPGHS-2 was incubated with [acetyl-C-14]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 PGHS-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 K-m value of the S516M mutant for arachidonate was 100 times that of native hPGHS-2. |
| Document Type: Article |
| Language: English |
Addresses:
1. MICHIGAN STATE UNIV, DEPT BIOCHEM, E LANSING, MI 48824 USA
2. VANDERBILT UNIV, DEPT BIOCHEM, NASHVILLE, TN 37232 USA |
| Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: NK184 |
| ISSN: 0021-9258 |
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