高等学校化学学报 ›› 2013, Vol. 34 ›› Issue (4): 875-880.doi: 10.7503/cjcu20120973

• 生物化学 • 上一篇    下一篇

氯过氧化物酶活性中心结构域中Mn2+与酶催化行为的关系

李海云1, 蒋育澄1,2, 胡满成1,2, 李淑妮1,2, 翟全国1,2   

  1. 1. 陕西师范大学化学化工学院;
    2. 大分子科学陕西省重点实验室, 西安 710062
  • 收稿日期:2012-10-26 出版日期:2013-04-10 发布日期:2013-03-21
  • 通讯作者: 蒋育澄,女,博士,教授,博士生导师,主要从事生物无机化学及酶工程方面的研究.E-mail:jyc@snnu.edu.cn E-mail:jyc@snnu.edu.cn
  • 基金资助:

    国家自然科学基金(批准号:21176150)资助.

Relationship Between Mn2+ in the Domain of Active Center of Chloroperoxidase and Enzyme Catalytic Performance

LI Hai-Yun1, JIANG Yu-Cheng1,2, HU Man-Cheng1,2, LI Shu-Ni1,2, ZHAI Quan-Guo1,2   

  1. 1. School of Chemistry & Chemical Engineering;
    2. Key Laboratory of Macromolecular Science of Shaanxi Province, Shaanxi Normal University, Xi'an 710062, China
  • Received:2012-10-26 Online:2013-04-10 Published:2013-03-21

摘要:

通过乙二胺四乙酸二钠(EDTA)配位竞争反应结合石墨炉原子吸收及电感耦合等离子体发射检测结果, 为氯过氧化物酶(CPO)活性中心血红素丙酸根与金属Mn2+配位结合提供了更直接的证据; 基于Mn2+移除前后CPO的紫外特征吸收光谱、 圆二色谱以及氯化活性和过氧化活性的变化, 阐明了Mn2+的存在对于保持酶活性中心的结构域、 稳定CPO的优势构象具有一定作用, 是酶分子表现活力所必需的结构元素. 本研究还发现移除Mn2+前后CPO血红素活性中心微环境的变化是一个可逆过程, 重新引入Mn2+后活性可恢复, 并且以外源性Ag+和Cr3+替代Mn2+后, CPO的氯化活性和过氧化活性分别比天然态的酶有所改善, 为通过化学修饰提高CPO的催化活性提供了新的途径.

关键词: 氯过氧化物酶, Mn2+, 酶催化行为, 化学修饰, 外源离子

Abstract:

The coordination of heme propionate with Mn2+ in active center of chloroperoxidase(CPO) was confirmed by means of removing the metal ions using ethylene diamine tetraacetic acid(EDTA) ligand competition reaction and determining Mn2+ by graphite furnace atomic absorption and inductively coupled plasmas atomic emissive spectrometry assay. The presence of Mn2+ was found playing a role in holding the active domain and maintaining preferential conformation of CPO based on the UV-Vis absorption spectroscopy, circular dichroism(CD) analysis as well as chlorination activity and peroxidation activity measurement before and after Mn2+ being removed from CPO. The maximum absorption at 398 nm of CPO, which displayed by iron(Ⅲ) porphyrin and named Soret band, decreased with a concomitant decrease of catalytic activity after Mn2+ in CPO being removed(MCPO). The analysis of CD spectra indicated that α-helix structure was uncoiling in MCPO. Furthermore, it was found that the changing of micro-environment around heme in CPO active site was a reversible process, and the catalytic activity may be recovered when Mn2+ was reintroduced, but it was a slow dynamic process that needed nearly 100 h. Moreover, the chlorination activity and peroxidation activity of CPO was enhanced compared with that of native CPO when exogenous Ag+ and Cr3+ are introduced instead of Mn2+. Especially, the introduction of Cr3+ could not only recover the peroxidation activity of MCPO but also elevate the relative activity to 106%. The chlorination activity of MCPO was also recovered and even enhanced by the introduing of Ag+. This process was related to the electron transfer involved in the enzymatic catalytic cycle.

Key words: Chloroperoxidase, Mn2+, Enzymatic performance, Chemical modification, Exogenous ion

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