Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry.
Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry.
复制标题
DOI：
10.1021/acs.biochem.5b01378
复制
发表时间：
2016-03-01
期刊：
影响因子：
2.9
通讯作者：
Malkowski MG
中科院分区：
文献类型：
--
作者：
Lucido MJ;Orlando BJ;Vecchio AJ;Malkowski MG
阿司匹林和其他非类固醇抗炎药以环氧合酶(COX-1和COX-2)为靶点，阻止前列腺素的形成。阿司匹林的独特之处在于，它通过乙酰化环氧合酶活性部位内的Ser-530来共价修饰每个酶。COX-1的乙酰化导致活性完全丧失，而COX-2的乙酰化导致单氧产物15(R)-羟基二十碳四烯酸(15R-HETE)的生成。Ser-530还被证明影响了前列腺素产品中氧气加成的立体化学。我们测定了S530T小鼠(MU)COX-2、阿司匹林乙酰化人(HU)COX-2和huCOX-2与水杨酸盐形成的络合物的晶体结构。结构揭示：1)乙酰化的Ser-530完全阻断了疏水沟槽的进入；2)观察到的水杨酸盐的结合姿势反映了乙酰化之前的酶-抑制物复合体；3)在S530T muCOX-2晶体结构中观察到的Thr-530旋转体没有阻碍疏水沟槽的进入。基于这些结构观察，结合对S530T/G533V双突变体的功能分析，我们提出了一个工作假说，即阿司匹林乙酰化的COX-2产生15R-HETE。我们还观察了在不同的洗涤剂体系和纳米盘中纯化的COX-2的乙酰化差异，表明在体外，洗涤剂和脂类结合酶的膜结合区域改变了乙酰化反应的速度。
Aspirin and other nonsterroidal anti-inflammatory drugs target the Cyclooxygenase enzymes (COX-1 and COX-2) to block the formation of prostaglandins. Aspirin is unique in that it covalently modifies each enzyme by acetylating Ser-530 within the cyclooxygenase active site. Acetylation of COX-1 leads to complete loss of activity, while acetylation of COX-2 results in the generation of the mono-oxygenated product 15(R)-hydroxyeicosatetraenoic acid (15R-HETE). Ser-530 has also been shown to influence the stereochemistry for oxygen addition into the prostaglandin product. We determined the crystal structures of S530T murine (mu) COX-2, aspirin-acetylated human (hu) COX-2, and huCOX-2 in complex with salicylate to 1.9Å, 2.0Å, and 2.4Å, respectively. The structures reveal that: 1) the acetylated Ser-530 completely blocks access to the hydrophobic groove; 2) the observed binding pose of salicylate is reflective of the enzyme-inhibitor complex prior to acetylation; and 3) the observed Thr-530 rotamer in the S530T muCOX-2 crystal structure does not impede access to the hydrophobic groove. Based on these structural observations, along with functional analysis of the S530T/G533V double mutant, we propose a working hypothesis for the generation of 15R-HETE by aspirin-acetylated COX-2. We also observe differential acetylation of COX-2 purified in various detergent systems and nanodiscs, indicating that detergent and lipid binding within the membrane-binding domain of the enzyme alters the rate of the acetylation reaction in vitro.