Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin.
Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin.
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DOI：
10.1016/s0021-9258(17)36820-5
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发表时间：
1994-05
期刊：
影响因子：
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通讯作者：
M. Lecomte;O. Laneuville;Chuan Ji;D L DeWitt;William L. Smith
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文献类型：
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作者：
M. Lecomte;O. Laneuville;Chuan Ji;D L DeWitt;William L. Smith
阿司匹林（乙酰水杨酸）处理cos-1细胞中表达的人（h）前列腺素内过氧化物H合酶（PGHS）-1导致加氧酶活性的时间依赖性失活。阿司匹林处理的hPGHS-2产生的酶保留加氧酶活性，但只形成15-羟基-5，8，11，13-二十碳四烯酸（15-HETE），而不是PGH 2。15-HETE是15 R配置的专有配置。天然和阿司匹林处理的hPGHS-2的花生四烯酸的Km值大致相同，表明花生四烯酸以类似的方式结合阿司匹林处理的和天然的hPGHS-2。如果如预期的那样，15 R-HETE的形成是通过从花生四烯酸中提取13 proS的氢来进行的，那么O2的插入必须发生在与氢提取相同的一侧;对于所有其他的脂氧合酶和环加氧酶，O2的添加是对面的。当微粒体hPGHS-2与[乙酰基-14 C]阿司匹林孵育时，酶被乙酰化。保留酶活性的hPGHS-2的S516 A突变体未被乙酰化。这表明Ser-516是hPGHS-2的阿司匹林乙酰化位点;该残基与PGHS-1的“活性位点”丝氨酸同源。hPGHS-2的S516 N突变体具有催化活性;相反，S516 Q突变体缺乏环氧合酶但保留过氧化物酶活性。因为在PGHS-1的情况下，较小的天冬酰胺取代足以消除环氧合酶活性，我们得出结论，PGHS-2的活性位点略大于PGHS-1。获得了hPGHS-2的S516 M突变体，其类似于阿司匹林乙酰化的hPGHS-2，因为该突变体使15 R-HETE作为其主要产物;然而，与阿司匹林乙酰化的hPGHS-2不同，S516 M突变体对花生四烯酸的Km值是天然hPGHS-2的100倍。
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.