Photoaffinity Labeling Reagents for Thermostable Diphosphate Synthase

在这项研究中，两种类型的异戊二烯基二磷酸酯的光活化类似物已被合成。一种类型是具有芳基叠氮化物基团作为可光活化基团的香叶基二磷酸类似物，另一种类型是含有可光活化的苯甲酰基苯氧基的异戊二烯基二磷酸类似物。这两种类型的类似物已被评估为类异戊二烯脱氢酶的底物和抑制剂，包括来自嗜热脂肪芽孢杆菌的热稳定法呢基二磷酸合酶（FPS）。放射性标记的类似物（在类似物的C-1位氢被^3H取代）。含有邻-叠氮基芳基的香叶基二磷酸类似物已被证明是竞争性抑制剂（Ki =10.5 μ M）。法呢基二磷酸合酶（FPS）经紫外灯照射后，酶活力完全丧失。另一方面，在不存在抑制剂的情况下未观察到活性损失。收集用放射性标记的抑制剂光标记的蛋白，并进行SDS-PAGE，表明产生放射性蛋白，并且PAGE上的条带出现在与野生型FPS几乎相同的位置。FPS、六异戊二烯基二磷酸合酶（HexPS）和十一异戊二烯基二磷酸合酶（UPS）。这些类似物进行扩链，将允许这些酶的疏水活性位点residuals.In结论，芳基叠氮类似物研究这里是有用的FPS作为探测工具和苯甲酰苯氧基类似物是有用的异戊二烯转移酶，产生较长的异戊二烯醇。

In this study, two types of photoactivatable analogs of isoprenyl diphosphates have been synthesized. One type is geranyl diphosphate analogs having arylazide group as photoactivatable group and other type is isoprenyl diphosphate analogs which contain photoacitivatable benzoylphenoxy group. The analogs of both types have been evaluated as substrates and inhibitors of isoprenoid synthases including thermostable farnesyl diphosphate synthase (FPS) from Bacillus stearothermophilus.. The radiolabeled analogs (substituted at the hydrogen of C-1 of analogs with ^3H). of both types were also synthesized.The geranyl diphosphate analogs containing rho-lazido aryl group has been shown to be competetive inhibitors (Ki =10.5 muM). When farnesyl diphosphate synthase (FPS) with the inhibitors was irradiated by UV-lamp, the complete loss of enzyme activity was observed. On the other hand, no loss of activity was observed in the absence of the inhibitor. The protein photolabeled with the radiolabeled inhibitor was collected and subjected on SDS-PAGE,insicating that the radioactive protein was produced and the band on PAGE appeared at the almost same position to that of wild FPS.The DMAPP and GPP analogs containing benzophenone as photoactivatable group were evalued as substrate and inhibitors three bacterial prenyl transferases : FPS,hexaprenyl diphosphate synthase (HexPS) and undecaprenyl diphoshate synthase (UPS). These analogs undergo chain extension and will allow identification of hydrophobic active site residues of these enzymes.In conclusion, aryl azide analog studied here is useful for FPS as probing tools and the analogs having benzoylphenoxy are useful for the prenyl transferases which produce longer prenols.