Structural effects of covalent inhibition of phospholipase A2 suggest allosteric coupling between membrane binding and catalytic sites.
Structural effects of covalent inhibition of phospholipase A2 suggest allosteric coupling between membrane binding and catalytic sites.
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磷脂酶 A2 共价抑制的结构效应表明膜结合和催化位点之间存在变构偶联。
DOI：
10.1016/s0006-3495(03)74985-6
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发表时间：
2003
影响因子：
3.4
通讯作者：
Tatulian,SurenA
中科院分区：
文献类型：
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作者：
Tatulian,SurenA
磷脂酶A2（PLA 2）结合到膜上并催化磷脂水解，从而启动脂质衍生的炎症介质的生物合成。通过共价修饰组氨酸48对溴苯甲酰甲基溴的催化作用，对蛇毒磷脂酶A2有完全的抑制作用。此外，His 48的修饰影响了PLA 2的结构、膜结合亲和力以及PLA 2对膜结构的影响。天然磷脂酶A2增加了流体膜的序参量，而凝胶态膜则相反。这些数据表明，膜脱水的PLA 2和PLA 2-膜氢键的形成。被抑制的PLA 2具有较低的膜结合亲和力，对膜水合作用和脂质序参量的影响较弱。虽然膜结合导致天然PLA 2中形成更灵活的α-螺旋，这对应于更快的酰胺氢交换，但修饰的酶对氢交换更具抗性，并且在膜结合时几乎没有结构变化。这些数据表明：1），修饰PLA 2的催化残基诱导构象变化，通过变构机制传播到膜结合表面; 2）天然PLA 2通过膜结合在界面活化过程中获得更多的动态特性; 3）抑制的PLA 2的整体构象，包括α-螺旋，不太稳定，不受膜结合的影响。这些研究结果提供了进一步的证据之间的膜结合（监管）网站和PLA 2的催化中心，这有助于界面激活的酶变构耦合。
Phospholipase A2(PLA2) binds to membranes and catalyzes phospholipid hydrolysis, thus initiating the biosynthesis of lipid-derived mediators of inflammation. A snake-venom PLA2was completely inhibited by covalent modification of the catalytic histidine 48 byp-bromophenacyl bromide. Moreover, His48modification affected PLA2structure, its membrane-binding affinity, and the effects of PLA2on the membrane structure. The native PLA2increased the order parameter of fluid membranes, whereas the opposite effect was observed for gel-state membranes. The data suggest membrane dehydration by PLA2and the formation of PLA2-membrane hydrogen bonding. The inhibited PLA2had lower membrane-binding affinity and exerted weaker effects on membrane hydration and on the lipid-order parameter. Although membrane binding resulted in formation of more flexibleα-helices in the native PLA2, which corresponds to faster amide hydrogen exchange, the modified enzyme was more resistant to hydrogen exchange and experienced little structural change upon membrane binding. The data suggest that 1), modification of a catalytic residue of PLA2induces conformational changes that propagate to the membrane-binding surface through an allosteric mechanism; 2), the native PLA2acquires more dynamic properties during interfacial activation via membrane binding; and 3), the global conformation of the inhibited PLA2, including theα-helices, is less stable and is not influenced by membrane binding. These findings provide further evidence for an allosteric coupling between the membrane-binding (regulatory) site and the catalytic center of PLA2, which contributes to the interfacial activation of the enzyme.