Identification of the electrophilic substrate-binding site of glutathione S-transferase P by photoaffinity labeling.
Identification of the electrophilic substrate-binding site of glutathione S-transferase P by photoaffinity labeling.
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通过光亲和标记鉴定谷胱甘肽 S-转移酶 P 的亲电子底物结合位点。
DOI：
10.1111/j.1432-1033.1995.tb20787.x
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Y. Hatanaka
中科院分区：
文献类型：
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作者：
J. Nishihira†;M. Sakai;S. Nishi;Y. Hatanaka
我们用光敏化合物S-[2-（2-氟-4-硝基苯氧基）乙基]谷胱甘肽进行光亲和标记，确定了大鼠谷胱甘肽S-转移酶P（GST-P）的亲电底物结合位点。这种光敏谷胱甘肽类似物抑制了对谷胱甘肽和1-氯-2，4-二硝基苯，一个假定的亲电底物的竞争性方式的催化活性。酶动力学研究表明，光活化谷胱甘肽类似物特异性结合在活性位点，活性位点由谷胱甘肽结合区（G-位点）和亲电子底物结合区（H-位点）组成。该方法包括以下步骤：S-[2-（2-氟-4-硝基苯氧基）乙基]谷胱甘肽与在大肠杆菌中表达的纯化的重组GST-P在冰上使用紫外线照射30分钟进行光化学反应。反应后，仅用谷胱甘肽固定化琼脂糖制备与谷胱甘肽类似物复合的GST-P。与类似物共价结合的GST-P用赖氨酰内肽酶（无色杆菌蛋白酶I）消化，并通过高效液相色谱法分离肽。通过肽图谱分析，仅观察到在340 nm处具有明显吸光度的单个主峰。收集肽并使用自动肽测序仪（ABI 477 A）进行分析。氨基酸序列分析表明，该肽由7个氨基酸残基组成，对应于GST-P的122-128位序列（Ala-Leu-Pro-Gly-Xaa-Leu-Lys）。在第五个循环中没有检测到明显的苯硫海因-氨基酸，这表明His 126被光敏谷胱甘肽类似物化学标记。结论His 126是GST-P亲电底物结合位点的氨基酸残基之一。
We determined the electrophilic substrate-binding site of rat glutathione S-transferase P (GST-P) by photoaffinity labeling using the photosensitive compound S-[2-(2-fluoro-4-nitrophenoxy)ethyl]glutathione. This photosensitive glutathione analogue inhibited the catalytic activity in a competitive manner against both glutathione and 1-chloro-2,4-dinitrobenzene, a putative electrophilic substrate. The enzyme kinetics indicated that the photoactivatable glutathione analogue was specifically bound at the active site, which consisted of glutathione-binding (G-site) and the electrophilic substrate-binding (H-site) regions. The procedure involved the following steps: S-[2-(2-fluoro-4-nitrophenoxy)ethyl]glutathione was photochemically reacted with a purified recombinant GST-P expressed in Escherichia coli using ultraviolet irradiation for 30 min on ice. After the reaction, only the GST-P complexed with the glutathione analogue was prepared with glutathione-immobilized agarose. The GST-P covalently bound with the analogue was digested with lysyl endopeptidase (Achromobacter protease I), and the peptides were separated by high-performance liquid chromatography. Only a single major peak with appreciable absorbance at 340 nm was observed by peptide mapping. The peptide was collected and analyzed using an automated peptide sequencer (ABI 477A). Amino acid sequence analysis showed that this peptide consisted of seven amino acid residues corresponding to the sequence at positions 122-128 of GST-P (Ala-Leu-Pro-Gly-Xaa-Leu-Lys). No appreciable phenylthiohydantoin-amino acid was detected at the fifth cycle, which indicated that His126 was chemically labeled with the photosensitive glutathione analogue. It was concluded that His126 was one of the amino acid residues forming the electrophilic substrate-binding site of GST-P.
使用光亲和标记定位两种大鼠肝脏谷胱甘肽 S-转移酶的部分活性位点。
DOI：
--
发表时间：
1989
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Hoesch,RM;Boyer,TD
通讯作者：
Boyer,TD