【摘要】：金屬蛋白大約能占到已知蛋白總數(shù)的百分之四十左右，它們參與一些基礎(chǔ)的生化反應(yīng)。金屬蛋白是利用金屬的氧化還原性質(zhì)和配位化學(xué)特性來(lái)實(shí)現(xiàn)其特定的生物學(xué)功能的，往往又伴隨著金屬離子周圍微弱的結(jié)構(gòu)變化。X射線吸收譜學(xué)(XAS)是一種具有亞原子分辨率的局域結(jié)構(gòu)研究方法，具有不受樣品形態(tài)限制、對(duì)金屬離子具有選擇性等突出優(yōu)點(diǎn)，是研究金屬蛋白中金屬活性中心精細(xì)結(jié)構(gòu)的理想技術(shù)。XAS方法在金屬蛋白結(jié)構(gòu)研究中的應(yīng)用也被專門命名為BioXAS。 蛋白質(zhì)的生物合成均起始于甲硫氨酸。在大多數(shù)情況下，N-端的甲硫氨酸在成熟蛋白中被去除。一般認(rèn)為只有在細(xì)菌中，N-端甲硫氨酸在蛋白合成之前被甲酰化。在翻譯后加工中，N-端甲硫氨酸先在去甲�；�(peptide deformylase，PDF)作用下被去甲�；趴梢员徽_切除。研究表明細(xì)菌中PDF的抑制會(huì)導(dǎo)致細(xì)菌的死亡，即PDF對(duì)于細(xì)菌存活是必需的。而在真核生物中，目前發(fā)現(xiàn)蛋白的翻譯后加工工程不涉及PDF的作用。因此，PDF是一個(gè)很好的藥物設(shè)計(jì)靶蛋白。 勾端螺旋體去甲酰化酶(Leptospira interrogans PDF)是一種重要的含鋅金屬蛋白酶，對(duì)鉤端螺旋體這一廣泛存在的致病菌的蛋白合成起著關(guān)鍵的催化作用。LiPDF酶促反應(yīng)受到反應(yīng)體系中多種因素尤其是pH值、溫度和各種離子的影響。當(dāng)pH值在7以下時(shí)，酶活很低；pH從7.5-10.5之間，酶活高而穩(wěn)定，即LiPDF有個(gè)相當(dāng)廣的最優(yōu)pH值，一直延伸到堿性區(qū)域。雖然pH對(duì)酶活的影響早已有之，但是關(guān)于pH對(duì)活性的影響并沒有圓滿的三維結(jié)構(gòu)上的解釋。 在E．coliPDF純化所制備出的不同金屬離子的樣品表明作為金屬酶，PDF的活性受到多種二價(jià)金屬離子如Co、Fe、Ni、Cu及Mg的影響。同樣，Co、Ni、Cu及Mg金屬離子溶液對(duì)LiPDF的活性產(chǎn)生了抑制作用，而Zn金屬離子溶液的存在對(duì)LiPDF的活性影響不大。Fe金屬離子溶液的存在雖然產(chǎn)生了一定效果的抑制作用，但相比較起其他金屬離子的作用而言，其活性表現(xiàn)出比較高的活性。雖然前人已經(jīng)報(bào)道了各種金屬離子形式的PDF研究，，但是關(guān)于金屬離子與活性的關(guān)系并沒有圓滿的三維結(jié)構(gòu)上的解釋。 本論文對(duì)LiPDF進(jìn)行了X射線譜學(xué)研究，包括一系列不同pH值的樣品的鋅K邊XANES譜，利用從頭計(jì)算(ab．initio)的多重散射(Multiple Scattering)方法確定金屬蛋白活性中心的精細(xì)結(jié)構(gòu)。比較這一系列不同pH值的活性中心地結(jié)構(gòu)，討論了其催化活性對(duì)pH值依賴性的原因，并給出合理的解釋。同時(shí)，用Co置換原生態(tài)蛋白中含有的Zn，研究其鈷K邊XANES譜并通過(guò)MXAN計(jì)算得到Co活性中心結(jié)構(gòu)。比較Zn和Co周圍活性中心結(jié)構(gòu)并對(duì)其金屬離子依賴性
[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
【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R377

【共引文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 袁旭;葉綠體核糖體蛋白PSRP-1部分cDNA序列的克隆[D];四川大學(xué);2001年

本文編號(hào)：2432720