通过快速定点突变表征人源含硒单链抗体酶的底物结合部位

高等学校化学学报 ›› 2010, Vol. 31 ›› Issue (3): 502.

通过快速定点突变表征人源含硒单链抗体酶的底物结合部位

宋健^1,2 , 徐俊杰^3,4 , 魏景艳³, 于扬³, 孙卫国³, 张桂荣²

1. 吉林大学电子科学与工程学院,
2. 白求恩医学院,
3. 药学院, 长春 130021;
4. 吉林医药学院基础医学院, 吉林 132013

收稿日期:2009-05-14 出版日期:2010-03-10 发布日期:2010-03-10
通讯作者: 张桂荣, 女, 博士, 教授, 主要从事疾病发生发展的分子机制及诊断、治疗方法的研究. E-mail: zhanggr@jlu.edu.cn
基金资助:
国家自然科学基金(批准号: 30870540)和吉林省科技发展计划项目(批准号: 20070726, 20070425, 200705368)资助.

Substrate-binding Sites of Human Selenium-containing Single Chain Abzyme by Quikchange Site-directed Mutagenesis

SONG Jian^1,2, XU Jun-Jie^3,4, WEI Jing-Yan³, YU Yang³, SUN Wei-Guo², ZHANG Gui-Rong^2*

1. College of Electronic Science and Engineering,
2. College of Fundamental Medicine,
3. College of Pharmaceutical Science; Jilin University, Changchun 130021, China;
4. Department of Biochemistry, School of Basic Medical Sciences, Jilin Medicine College, Jilin 132013, China

Received:2009-05-14 Online:2010-03-10 Published:2010-03-10
Contact: ZHANG Gui-Rong. E-mail: zhanggr@jlu.edu.cn
Supported by:
国家自然科学基金(批准号: 30870540)和吉林省科技发展计划项目(批准号: 20070726, 20070425, 200705368)资助.

摘要/Abstract

摘要：

为了对人源含硒单链抗体酶Se-scFv-B3的底物结合部位和催化基团进行研究, 在理论预测的基础上, 通过快速定点突变法分别在2个理论预测的底物结合部位(位点1和位点2)内选定Ala180和Ala44定点突变为丝氨酸(Ser). 2个突变体蛋白经化学修饰将Ser转变成谷胱甘肽过氧化物酶(GPX)的催化基团硒代半胱氨酸(Sec)后, 前者的GPX活力达到了Se-scFv-B3的2倍多, 而后者的GPX活力没有明显提高, 这表明位点1可能是主要的底物结合部位, 与理论预测的结果一致.

关键词: 快速定点突变; 人源含硒单链抗体酶; 底物结合部位

Abstract:

Ala180 in site 1 and Ala44 in site 2 of human selenium-containing single chain abzyme Se-scFv-B3 were chosen to be mutated to serines, respectively, to study the substrate binding sites and the catalytic group of Se-scFv-B3 and to find the reason why the Se-scFv-B3 has lower catalytic activity based on the theoretical anticipation for the protein. After serine residue was reacted into secystins by chemical modification, the activity of the anterior one is about two times than that of the Se-scFv-B3, but the latter one did not offer a significant improvement. This indicated that site 1 was the predominant binding site for GSH, which was consistent with the foregoing conjecture and the results of energy calculation. In this study, the GPX activity of Se-scFv-B3 was improved and the site 1 preliminarily confirmed to be the substrate binding site based on both theoretical and experimental research. All of these observations will be useful for further work in this area.

Key words: Quikchange site-directed mutagenesis; Human selenium-containing sigle chain abzyme; Substrate-binding site

TrendMD:

宋健, 徐俊杰, 魏景艳, 于扬, 孙卫国, 张桂荣. 通过快速定点突变表征人源含硒单链抗体酶的底物结合部位. 高等学校化学学报, 2010, 31(3): 502.

SONG Jian, XU Jun-Jie, WEI Jing-Yan, YU Yang, SUN Wei-Guo, ZHANG Gui-Rong. Substrate-binding Sites of Human Selenium-containing Single Chain Abzyme by Quikchange Site-directed Mutagenesis. Chem. J. Chinese Universities, 2010, 31(3): 502.

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