Mancini J A, Vickers P J, O'Neill G P, Boily C, Falgueyret J P, Riendeau D
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Québec, Canada.
Mol Pharmacol. 1997 Jan;51(1):52-60. doi: 10.1124/mol.51.1.52.
Aspirin (ASA) acetylates Ser516 of prostaglandin G/H synthase-2 (PGHS-2) resulting in a modified enzyme that converts arachidonic acid to 15(R)-hydroxy-eicosatetraeroic acid [15(R)-HETE]. ASA has pharmacological benefits that may not all be limited to inhibition of prostaglandin synthesis, and this study was initiated to further investigate the properties of ASA-acetylated PGHS-2 and of the mutation of Ser516 to methionine, which mimics ASA acetylation. Both the S516M mutant and ASA-acetylated form of PGHS-2 (ASA-PGHS-2) synthesize 15(R)-HETE and have apparent K(m) values for arachidonic acid within 10-fold of the apparent K(m) value for untreated PGHS-2. The time courses of turnover-dependent inactivation were similar for reactions catalyzed by PGHS-2 and ASA-PGHS-2, whereas the PGHS-2(S516M) showed a decrease in both the initial rate of 15-HETE production and rate of enzyme inactivation. The production of 15-HETE by modified PGHS-2 was sensitive to inhibition by most nonsteroidal anti-inflammatory drugs (NSAIDs), including selective PGHS-2 inhibitors. As observed for the cyclooxygenase activity of PGHS-2, the inhibition of 15-HETE production by indomethacin was time-dependent for both ASA-PGHS-2 and PGHS-2(S516M). However, two potent, structurally related NSAIDs, diclofenac and meclofenamic acid, do not inhibit either ASA-PGHS-2 or the PGHS-2(S516M) mutant. These results demonstrate that the sensitivity to inhibition by NSAIDs of the 15-HETE production by ASA-treated PGHS-2 is different than that of prostaglandin production by PGHS-2 and that Ser516 plays an important role in the interaction with fenamate inhibitors. The results also indicate that the conversion of arachidonic acid to 15-HETE by ASA-PGHS-2 is an efficient process providing a unique mechanism among NSAIDs that will not lead to arachidonic acid accumulation or shunting to other biosynthetic pathways.
阿司匹林（ASA）使前列腺素G/H合酶-2（PGHS-2）的Ser516位点乙酰化，产生一种修饰后的酶，该酶可将花生四烯酸转化为15（R）-羟基二十碳四烯酸[15（R）-HETE]。ASA具有的药理益处可能并非都局限于抑制前列腺素合成，开展本研究是为了进一步探究ASA乙酰化的PGHS-2以及将Ser516突变为甲硫氨酸（模拟ASA乙酰化）后的特性。PGHS-2的S516M突变体和ASA乙酰化形式（ASA-PGHS-2）均能合成15（R）-HETE，且花生四烯酸的表观K（m）值与未处理的PGHS-2的表观K（m）值相差在10倍以内。PGHS-2和ASA-PGHS-2催化的反应中，与周转相关的失活时间进程相似，而PGHS-2（S516M）在15-HETE产生的初始速率和酶失活速率上均有所降低。修饰后的PGHS-2产生15-HETE的过程对大多数非甾体抗炎药（NSAIDs）敏感，包括选择性PGHS-2抑制剂。正如在PGHS-2的环氧化酶活性中观察到的那样，吲哚美辛对ASA-PGHS-2和PGHS-2（S516M）的15-HETE产生的抑制作用均呈时间依赖性。然而，两种强效的、结构相关的NSAIDs，双氯芬酸和甲氯芬那酸，对ASA-PGHS-2或PGHS-2（S516M）突变体均无抑制作用。这些结果表明，ASA处理的PGHS-2产生15-HETE对NSAIDs抑制作用的敏感性与PGHS-2产生前列腺素的敏感性不同，且Ser516在与芬那酸抑制剂的相互作用中起重要作用。结果还表明，ASA-PGHS-2将花生四烯酸转化为15-HETE是一个高效过程，在NSAIDs中提供了一种独特机制，不会导致花生四烯酸积累或转向其他生物合成途径。
