钩端螺旋体去甲酰化酶的X射线吸收谱学研究
发布时间：2019-03-01 19:12
【摘要】：金属蛋白大约能占到已知蛋白总数的百分之四十左右，它们参与一些基础的生化反应。金属蛋白是利用金属的氧化还原性质和配位化学特性来实现其特定的生物学功能的，往往又伴随着金属离子周围微弱的结构变化。X射线吸收谱学(XAS)是一种具有亚原子分辨率的局域结构研究方法，具有不受样品形态限制、对金属离子具有选择性等突出优点，是研究金属蛋白中金属活性中心精细结构的理想技术。XAS方法在金属蛋白结构研究中的应用也被专门命名为BioXAS。 蛋白质的生物合成均起始于甲硫氨酸。在大多数情况下，N-端的甲硫氨酸在成熟蛋白中被去除。一般认为只有在细菌中，N-端甲硫氨酸在蛋白合成之前被甲酰化。在翻译后加工中，N-端甲硫氨酸先在去甲酰化酶(peptide deformylase，PDF)作用下被去甲酰化，才可以被正确切除。研究表明细菌中PDF的抑制会导致细菌的死亡，即PDF对于细菌存活是必需的。而在真核生物中，目前发现蛋白的翻译后加工工程不涉及PDF的作用。因此，PDF是一个很好的药物设计靶蛋白。 勾端螺旋体去甲酰化酶(Leptospira interrogans PDF)是一种重要的含锌金属蛋白酶，对钩端螺旋体这一广泛存在的致病菌的蛋白合成起着关键的催化作用。LiPDF酶促反应受到反应体系中多种因素尤其是pH值、温度和各种离子的影响。当pH值在7以下时，酶活很低；pH从7.5-10.5之间，酶活高而稳定，即LiPDF有个相当广的最优pH值，一直延伸到碱性区域。虽然pH对酶活的影响早已有之，但是关于pH对活性的影响并没有圆满的三维结构上的解释。 在E．coliPDF纯化所制备出的不同金属离子的样品表明作为金属酶，PDF的活性受到多种二价金属离子如Co、Fe、Ni、Cu及Mg的影响。同样，Co、Ni、Cu及Mg金属离子溶液对LiPDF的活性产生了抑制作用，而Zn金属离子溶液的存在对LiPDF的活性影响不大。Fe金属离子溶液的存在虽然产生了一定效果的抑制作用，但相比较起其他金属离子的作用而言，其活性表现出比较高的活性。虽然前人已经报道了各种金属离子形式的PDF研究，，但是关于金属离子与活性的关系并没有圆满的三维结构上的解释。 本论文对LiPDF进行了X射线谱学研究，包括一系列不同pH值的样品的锌K边XANES谱，利用从头计算(ab．initio)的多重散射(Multiple Scattering)方法确定金属蛋白活性中心的精细结构。比较这一系列不同pH值的活性中心地结构，讨论了其催化活性对pH值依赖性的原因，并给出合理的解释。同时，用Co置换原生态蛋白中含有的Zn，研究其钴K边XANES谱并通过MXAN计算得到Co活性中心结构。比较Zn和Co周围活性中心结构并对其金属离子依赖性
[Abstract]:Metalloproteins can account for about 40% of the total number of known proteins, and they are involved in some basic biochemical reactions. Metalloproteins make use of the redox and coordination chemical properties of metals to achieve their specific biological functions. X-ray absorption spectroscopy (XAS) is a local structure research method with sub-atomic resolution, which is not limited by the shape of the sample. It is an ideal technique to study the fine structure of metal active center in metalprotein due to its selectivity to metal ions. The application of XAS method in the study of metalprotein structure is also named BioXAS.. The biosynthesis of proteins begins with methionine. In most cases, N-terminal methionine is removed from mature proteins. It is generally believed that N-terminal methionine is formylated only in bacteria before protein synthesis. In post-translation processing, N-terminal methionine can be removed correctly only if it is decarbonylated under the action of de-formyl acylase (peptide deformylase,PDF). Studies have shown that inhibition of PDF in bacteria can lead to bacterial death, that is, PDF is necessary for bacterial survival. In eukaryotes, it has been found that post-translation processing of proteins does not involve the role of PDF. Therefore, PDF is a good drug design target protein. Leptospira norformylase (Leptospira interrogans PDF) is an important zinc-containing metalloproteinases. It plays a key role in the protein synthesis of leptospira, a widespread pathogen. LiPDF enzymatic reaction is influenced by many factors, such as pH value, temperature and various ions in the reaction system. When the pH value is below 7, the enzyme activity is very low, and the pH is between 7.5 and 10.5.The enzyme activity is high and stable, that is, LiPDF has a very wide optimal pH value, which extends to the alkaline region. Although the effect of pH on enzyme activity has long existed, there is no satisfactory three-dimensional structure explanation for the effect of pH on enzyme activity. The samples of different metal ions prepared by E.coliPDF purification show that the activity of PDF is affected by a variety of divalent metal ions such as Co,Fe,Ni,Cu and Mg as metalases. Similarly, Co,Ni,Cu and Mg metal ion solution inhibited the activity of LiPDF, but the presence of Zn metal ion solution had little effect on the activity of LiPDF. Although the existence of Fe metal ion solution had a certain effect on the activity of LiPDF, the existence of Fe metal ion solution had a certain inhibitory effect on the activity of LiPDF. However, compared with other metal ions, the activity of these ions is higher than that of other metal ions. Although PDF studies of various metal ion forms have been reported, there is no satisfactory three-dimensional structure explanation for the relationship between metal ions and activity. In this paper, LiPDF was studied by X-ray spectroscopy, including the Zn-K-edge XANES spectra of a series of samples with different pH values. The fine structure of the active center of metalloprotein was determined by ab initio (ab.initio) multiple scattering (Multiple Scattering) method. The structure of the active centers with different pH values was compared, and the reasons for the dependence of the catalytic activity on the pH values were discussed, and a reasonable explanation was given. At the same time, the Cobalt K-edge XANES spectra were studied by using Co to replace the Zn, contained in the proto-eco-protein, and the active center structure of Co was obtained by MXAN calculation. Comparison of the structures of active centers around Zn and Co and their dependence on metal ions
【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R377
本文编号：2432720
[Abstract]:Metalloproteins can account for about 40% of the total number of known proteins, and they are involved in some basic biochemical reactions. Metalloproteins make use of the redox and coordination chemical properties of metals to achieve their specific biological functions. X-ray absorption spectroscopy (XAS) is a local structure research method with sub-atomic resolution, which is not limited by the shape of the sample. It is an ideal technique to study the fine structure of metal active center in metalprotein due to its selectivity to metal ions. The application of XAS method in the study of metalprotein structure is also named BioXAS.. The biosynthesis of proteins begins with methionine. In most cases, N-terminal methionine is removed from mature proteins. It is generally believed that N-terminal methionine is formylated only in bacteria before protein synthesis. In post-translation processing, N-terminal methionine can be removed correctly only if it is decarbonylated under the action of de-formyl acylase (peptide deformylase,PDF). Studies have shown that inhibition of PDF in bacteria can lead to bacterial death, that is, PDF is necessary for bacterial survival. In eukaryotes, it has been found that post-translation processing of proteins does not involve the role of PDF. Therefore, PDF is a good drug design target protein. Leptospira norformylase (Leptospira interrogans PDF) is an important zinc-containing metalloproteinases. It plays a key role in the protein synthesis of leptospira, a widespread pathogen. LiPDF enzymatic reaction is influenced by many factors, such as pH value, temperature and various ions in the reaction system. When the pH value is below 7, the enzyme activity is very low, and the pH is between 7.5 and 10.5.The enzyme activity is high and stable, that is, LiPDF has a very wide optimal pH value, which extends to the alkaline region. Although the effect of pH on enzyme activity has long existed, there is no satisfactory three-dimensional structure explanation for the effect of pH on enzyme activity. The samples of different metal ions prepared by E.coliPDF purification show that the activity of PDF is affected by a variety of divalent metal ions such as Co,Fe,Ni,Cu and Mg as metalases. Similarly, Co,Ni,Cu and Mg metal ion solution inhibited the activity of LiPDF, but the presence of Zn metal ion solution had little effect on the activity of LiPDF. Although the existence of Fe metal ion solution had a certain effect on the activity of LiPDF, the existence of Fe metal ion solution had a certain inhibitory effect on the activity of LiPDF. However, compared with other metal ions, the activity of these ions is higher than that of other metal ions. Although PDF studies of various metal ion forms have been reported, there is no satisfactory three-dimensional structure explanation for the relationship between metal ions and activity. In this paper, LiPDF was studied by X-ray spectroscopy, including the Zn-K-edge XANES spectra of a series of samples with different pH values. The fine structure of the active center of metalloprotein was determined by ab initio (ab.initio) multiple scattering (Multiple Scattering) method. The structure of the active centers with different pH values was compared, and the reasons for the dependence of the catalytic activity on the pH values were discussed, and a reasonable explanation was given. At the same time, the Cobalt K-edge XANES spectra were studied by using Co to replace the Zn, contained in the proto-eco-protein, and the active center structure of Co was obtained by MXAN calculation. Comparison of the structures of active centers around Zn and Co and their dependence on metal ions
【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R377
【共引文献】
相关硕士学位论文 前1条
1 袁旭;叶绿体核糖体蛋白PSRP-1部分cDNA序列的克隆[D];四川大学;2001年
本文编号：2432720
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