高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (1): 146.doi: 10.7503/cjcu20130792

• 物理化学 • 上一篇    下一篇

嗜热蛋白酶PH1704别构中心量子化学计算与突变体动力学

詹冬玲1,2, 高楠3, 韩葳葳1(), 冯雁1()   

  1. 1. 吉林大学分子酶学工程教育部重点实验室, 长春 130012
    2. 吉林农业大学食品科学与工程学院, 长春 130118
    3. 中国科学院长春应用化学研究所, 长春 130022
  • 收稿日期:2013-08-15 出版日期:2014-01-10 发布日期:2013-12-13
  • 作者简介:联系人简介: 冯 雁, 女, 博士, 教授, 博士生导师, 主要从事生物化学与分子生物学研究. E-mail:yfeng@jlu.edu.cn;韩葳葳, 女, 博士, 副教授, 主要从事计算生物学研究. E-mail:weiweihan@jlu.edu.cn
  • 基金资助:
    国家“九七三”计划项目(批准号: 2012CB721003)、 国家自然科学基金(批准号: 31070638)和吉林省自然科学基金(批准号:201015109)资助

Quantum Chemistry Calculation of Thermophilic Protease PH1704 Allosteric Center and Mutant Dynamics

ZHAN Dongling1,2, GAO Nan3, HAN Weiwei1,*(), FENG Yan1,*()   

  1. 1. Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education,Jilin University, Changchun 130012, China
    2. College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China
    3. Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,Changchun 130022, China
  • Received:2013-08-15 Online:2014-01-10 Published:2013-12-13
  • Contact: HAN Weiwei,FENG Yan E-mail:weiweihan@jlu.edu.cn;yfeng@jlu.edu.cn
  • Supported by:
    † Supported by the National Basic Research Program of China(No.2012CB721003), the National Natural Science Foundation of China(No.31070638) and the Natural Science Foundation of Jilin Province, China(No.201015109)

摘要:

通过量子化学计算, 确定嗜热菌Pyrococcus horikoshii OT3的PH1704蛋白酶别构位点的关键残基为Arg113, Tyr120和Asn129. 其中, Arg113及Asn129与别构抑制剂结合, 参与别构调控. Tyr120残基位于亚基交界面附近, 并与亲核残基Cys100之间以氢键相连, 可通过影响亚基聚合来影响酶的亲核催化. DJ-1超家族的4种构建蛋白的结构显示, 120位点位于亚基交界面处, 影响亚基的聚合, 进而影响蛋白酶的活力, 并间接参与别构调控. 分子生物学实验显示, 突变体R113T/Y120P/N129D的kcat/km(L·μmol-1·min-1)值是野生型kcat/km值的6倍, h系数由野生型的0.86转变为1.3, 负协同效应消失. 113和129位点处阴离子别构剂脱离, 从而破坏113, 120和129位点间的封闭环结构, 使AC交界面α7螺旋(124~129, 524~529)间聚合度增强; 120位点残基由Tyr转变为Pro, 与Cys100间氢键断裂, 亲核进攻的阻力减小, 从而使酶活力提高, 别构负调控消失.

关键词: 嗜热蛋白酶, 量子化学计算, 别构中心, 定点突变

Abstract:

The PH1704 allosteric sites were studied with the quantum chemistry analysis and the crystal structure analysis. The results show that key residues are Arg113, Tyr120 and Asn129. Tyr120 is connected with nucleophilic residues Cys100 by a hydrogen bond, participates in enzyme nucleophilic catalyst, and is validated by fixed-point mutation of molecular biology experiments. The structures of four building protein of DJ-1 superfamily show that the 120 site locates in the substrate binding pocket in the subunit interface and affects the enzyme activity of the protein. The kcat/km(L·μmol-1·min-1) value of mutant R113T/Y120P/N129D is six times higher than that of the wild-type and the Hill coefficient changes from 0.86(wild type) to 1.3 with negative cooperativity disappearing. The main reason is that the residue of 120 site changes from Tyr to Pro, and the hydrogen bonds between Tyr120 and Cys100 are broken, thus its nucleophilic attacking resis-tance decreases, which causes the enzyme activity to increase. The mutations of 113 and 129 sites lead to the detachment of the anionic allosteric agent, thus the negative cooperativity disappears. This work predictes the allosteric site of thermophilic protease by quantum chemistry and crystal structure analysis and provides a solid foundation for further research on the allosteric enzyme of DJ-1 superfamily.

Key words: Thermophilic protease, Quantum chemistry calculation, Allosteric center, Site-mutant

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