Spatial requirements for 15-(R)-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid synthesis within the cyclooxygenase active site of murine COX-2 -: Why acetylated COX-1 does not synthesize 15-(R)-HETE
Spatial requirements for 15-(R)-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid synthesis within the cyclooxygenase active site of murine COX-2 -: Why acetylated COX-1 does not synthesize 15-(R)-HETE
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DOI：
10.1074/jbc.275.9.6586
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发表时间：
2000-03-03
影响因子：
4.8
通讯作者：
Marnett, LJ
中科院分区：
文献类型：
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作者：
Rowlinson, SW;Crews, BC;Marnett, LJ
环加氧酶的两个同工异构体COX-1和COX-2分别在环加氧酶活性位点Ser-530和Ser-516处被阿司匹林乙酰化。乙酰化的COX-2本质上是一种脂氧合酶，可生成15-(R)-羟二糖四烯酸（18-HETE）和11-(R)-羟二糖四烯酸（11- hete），而乙酰化的COX-1不能氧化花生四烯酸生成任何产物。由于COX异构体在结构上相似，并且具有大约60%的氨基酸同源性，我们假设环加氧酶活性位点的差异必须解释乙酰化COX-1无法生成11-和15-HETE的原因。通过比较COX-1和COX -2的晶体结构，预测了残基Val-434、Arg-513和Val-523，以解释在COX异构体之间观察到的空间和柔韧性差异。将小鼠COX-2中的Val-434、Arg-513和Val-523位点定向突变为其cox - 1等效物，导致阿司匹林治疗后11-和15-HETE的产生消失，证实了这些残基是调节HETE产生的主要同种异构体选择性决定因素的假设。经阿司匹林处理的R513H mCOX-2产生15-HETE的能力，尽管数量减少，表明该残基不是花生四烯酸羧酸盐的替代结合位点，也不是调节HETE产生的唯一特异性决定因素。进一步的实验确定Ser-530上乙酰基部分赋予的空间体积是否阻止了花生四烯酸的ω -端在mCOX-2的顶部通道腔内结合。V228F和G533A都产生了野生型样的产物，但经过乙酰化后，两者都不能产生HETE产物。这表明花生四烯酸在前列腺素和HETE产物的生产中以l形结合构型定向。
The two isoforms of cyclooxygenase, COX-1 and COX-2, are acetylated by aspirin at Ser-530 and Ser-516, respectively, in the cyclooxygenase active site. Acetylated COX-2 is essentially a lipoxygenase, making 15-(R)-hydroxyeicosatetraenoic acid (18-HETE) and 11-(R)-hydroxyeicosatetraenoic acid (11-HETE), whereas acetylated COX-1 is unable to oxidize arachidonic acid to any products. Because the COX isoforms are structurally similar and share approximately 60% amino acid identity, we postulated that differences within the cyclooxygenase active sites must account for the inability of acetylated COX-1 to make 11- and 15-HETE. Residues Val-434, Arg-513, and Val-523 were predicted by comparison of the COX-1 and -2 crystal structures to account for spatial and flexibility differences observed ed between the COX isoforms. Site-directed mutagenesis of Val-434, Arg-513, and Val-523 in mouse COX-2 to their COX-I equivalents resulted in abrogation of 11- and 15-HETE production after aspirin treatment, confirming the hypothesis that these residues are the major isoform selectivity determinants regulating HETE production. The ability of aspirin-treated R513H mCOX-2 to make 15-HETE, although in reduced amounts, indicates that this residue is not an alternate binding site for the carboxylate of arachidonate and that it is not the only specificity determinant regulating HETE production. Further experiments were undertaken to ascertain whether the steric bulk imparted by the acetyl moiety on Ser-530 prevented the omega-end of arachidonic acid from binding within the top channel cavity in mCOX-2. Site directed mutagenesis was performed to change Val-228, which resides at the junction of the main cyclooxygenase channel and the top channel, and Gly-533, which is in the top channel, Both V228F and G533A produced wild type-like product profiles, but, upon acetylation, neither was able to make HETE products. This suggests that arachidonic acid orientates in a L-shaped binding configuration in the production of both prostaglandin and HETE products.