Forced evolution of glutathione S-transferase to create a more efficient drug detoxication enzyme.
Forced evolution of glutathione S-transferase to create a more efficient drug detoxication enzyme.
复制标题
强制进化谷胱甘肽S-转移酶以创造更有效的药物解毒酶。
DOI：
10.1073/pnas.92.18.8140
复制
发表时间：
1995
影响因子：
11.1
通讯作者：
Fahl,WE
中科院分区：
文献类型：
--
作者：
Gulick,AM;Fahl,WE
哺乳动物细胞中的谷胱甘肽 S-转移酶 (EC 2.5.1.18) 催化结合，从而催化结构多样的亲电子环境致癌物和烷化药物（包括抗肿瘤氮芥）的解毒。我们提出，非特异性亲电子结合位点的结构改变将产生突变酶，其对单一药物的解毒效率提高，并且这些突变体可以作为有用的体细胞转基因来保护健康人类细胞免受癌症化疗方案中使用的单一烷化剂的侵害。对共同组成谷胱甘肽 S-转移酶亲电子结合位点的三个区域（残基 9-14、102-112 和 210-220）进行随机诱变，然后选择表达具有氮芥氮芥（20-500 µM）的酶文库的大肠杆菌，产生了催化效率显着提高的突变酶。 氮芥结合（kcat 增加高达 15 倍，kcat/Km 增加高达 6 倍），并赋予高达 31 倍的耐药性，比野生型酶所赋予的耐药性高 9 倍。结果提出了修饰药物和致癌物代谢酶的一般策略，以在原核和真核植物和动物细胞中实现所需的耐药性。
Glutathione S-transferases (EC 2.5.1.18) in mammalian cells catalyze the conjugation, and thus, the detoxication of a structurally diverse group of electrophilic environmental carcinogens and alkylating drugs, including the antineoplastic nitrogen mustards. We proposed that structural alteration of the nonspecific electrophile-binding site would produce mutant enzymes with increased efficiency for detoxication of a single drug and that these mutants could serve as useful somatic transgenes to protect healthy human cells against single alkylating agents used in cancer chemotherapy protocols. Random mutagenesis of three regions (residues 9-14, 102-112, and 210-220), which together compose the glutathione S-transferase electrophile-binding site, followed by selection of Escherichia coli expressing the enzyme library with the nitrogen mustard mechlorethamine (20-500 microM), yielded mutant enzymes that showed significant improvement in catalytic efficiency for mechlorethamine conjugation (up to 15-fold increase in kcat and up to 6-fold increase in kcat/Km) and that confer up to 31-fold resistance, which is 9-fold greater drug resistance than that conferred by the wild-type enzyme. The results suggest a general strategy for modification of drug- and carcinogen-metabolizing enzymes to achieve desired resistance in both prokaryotic and eukaryotic plant and animal cells.