Investigation of the binding of isoform-selective inhibitors to prostaglandin endoperoxide synthases using fluorescence spectroscopy.
Investigation of the binding of isoform-selective inhibitors to prostaglandin endoperoxide synthases using fluorescence spectroscopy.
复制标题
使用荧光光谱研究异构体选择性抑制剂与前列腺素内过氧化物合酶的结合。
DOI：
10.1021/bi971691n
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Marnett,LJ
中科院分区：
文献类型：
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作者：
Lanzo,CA;Beechem,JM;Talley,J;Marnett,LJ
前列腺素内过氧化物合酶 (PGHS) 是一种血红素蛋白，可催化前列腺素和血栓素生物合成的关键步骤。 PGHS 存在两种亚型，一种称为 PGHS-1 的组成型，一种称为 PGHS-2 的诱导型。我们在此报告同种型选择性抑制剂与 PGHS-1 和 PGHS-2 相互作用的荧光共振能量转移分析。通过测量抑制剂荧光能量转移至 PGHS 血红素辅基导致的荧光猝灭，我们确定这些抑制剂结合在花生四烯酸底物进入通道中，PGHS-1 与 PGHS-1 抑制剂的 R0 为 35 Å，PGHS-2 与 PGHS-2 抑制剂的 R0 为 21 Å。观察到的荧光猝灭是完全动态的，主要是血红素的猝灭。时间分辨结果与分子模型相结合，确定抑制剂到血红素部分的距离在 PGHS-1 中为 20 Å，在 PGHS-2 中为 18 Å。初步的停流动力学研究表明，猝灭速率受到一级蛋白质转变的限制，该转变很慢，并且结合的抑制剂经历快速交换。
Prostaglandin endoperoxide synthase (PGHS) is a heme protein that catalyzes the committed step in prostaglandin and thromboxane biosynthesis. Two isoforms of PGHS exist, a constitutive form termed PGHS-1 and an inducible form termed PGHS-2. We report here fluorescence resonance energy transfer analysis of isoform-selective inhibitors interacting with PGHS-1 and PGHS-2. By measuring fluorescence quenching due to the energy transfer of the inhibitor fluorescence to the heme prosthetic group of PGHS, we determined these inhibitors bind in the arachidonic acid substrate access channel with anR0of 35 Å for PGHS-1 with the PGHS-1 inhibitor and anR0of 21 Å for PGHS-2 with the PGHS-2 inhibitor. The observed fluorescence quenching is completely dynamic and dominated by quenching by the heme. Time-resolved results combined with molecular modeling determine the distance from the inhibitor to the heme moiety to be 20 Å in PGHS-1 and 18 Å in PGHS-2. Preliminary stopped-flow kinetic studies reveal that the rate of quenching is limited by a first-order protein transition, which is slow, and that bound inhibitor undergoes rapid exchange.