Oakley A J, Rossjohn J, Lo Bello M, Caccuri A M, Federici G, Parker M W
Ian Porter Foundation Protein Crystallography Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
Biochemistry. 1997 Jan 21;36(3):576-85. doi: 10.1021/bi962316i.
The potent diuretic drug ethacrynic acid has been tested in clinical trials as an adjuvant in chemotherapy. Its target is the detoxifying enzyme glutathione transferase which is often found overexpressed in cancer tissues. We have solved the crystal structures of human pi class glutathione transferase P1-1 in complex with the inhibitor ethacrynic acid and its glutathione conjugate. Ethacrynic acid is found to bind in a nonproductive mode to one of the ligand binding sites of the enzyme (the H site) while the glutathione binding site (G site) is occupied by solvent molecules. There are no structural rearrangements of the G site in the absence of ligand. The structure indicates that bound glutathione is required for ethacrynic acid to dock into the H site in a productive binding mode. The binding of the ethacrynic acid-glutathione conjugate shows that the contacts of the glutathione moiety with the protein are identical to those observed in crystal structures of the enzyme with other glutathione-based substrates and inhibitors. The ethacrynic acid moiety of the conjugate binds in the H site in a fashion that has not been observed in crystal structures of other glutathione-based inhibitor complexes. The crystal structures implicate Tyr 108 as an electrophilic participant in the Michael addition of glutathione to ethacrynic acid.
强效利尿药依他尼酸已在临床试验中作为化疗辅助药物进行了测试。其靶点是解毒酶谷胱甘肽转移酶，该酶在癌组织中常过度表达。我们解析了人π类谷胱甘肽转移酶P1-1与抑制剂依他尼酸及其谷胱甘肽共轭物复合物的晶体结构。发现依他尼酸以非有效模式结合到酶的一个配体结合位点（H位点），而谷胱甘肽结合位点（G位点）被溶剂分子占据。在没有配体的情况下，G位点没有结构重排。该结构表明，结合的谷胱甘肽是依他尼酸以有效结合模式对接至H位点所必需的。依他尼酸-谷胱甘肽共轭物的结合表明，谷胱甘肽部分与蛋白质的接触与在该酶与其他基于谷胱甘肽的底物和抑制剂的晶体结构中观察到的接触相同。共轭物的依他尼酸部分以一种在其他基于谷胱甘肽的抑制剂复合物的晶体结构中未观察到的方式结合在H位点。晶体结构表明Tyr 108是谷胱甘肽与依他尼酸进行迈克尔加成反应中的亲电参与者。
