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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 林俊宏 | |
dc.contributor.author | Yu-Nong Lin | en |
dc.contributor.author | 林宥穠 | zh_TW |
dc.date.accessioned | 2021-06-13T06:52:33Z | - |
dc.date.available | 2007-07-30 | |
dc.date.copyright | 2005-07-30 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-07-28 | |
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(2001) Triclosan offers protection against blood stages of malaria by inhibiting enoyl-ACP reductase of Plasmodium falciparum. Nat. Med. 7, 167–173. Sweeney, A. M., Lange, R. L., Fernandes, R. P. M., Schulz, H., Dale, G. E., Douangamath, A., Proteau, P. J., Oefner, C. (2005) The crystal structure of E.coli 1-deoxy-D-xylulose-5-phosphate reductoisomerase in a ternary complex with the antimalarial compound fosmidomycin and NADPH reveals a tight-binding closed enzyme conformation. J. Mol. Biol. 345, 115–127. Takahashi, S., Kuzuyama, T., Watanabe, H., Seto, H. (1998). A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-Derythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis. Proc. Natl Acad. Sci. USA, 95, 9879–9884. Tulsiani, D. R. P., Skudlarek, M. D., Orgebin-Crist, M. C. (1989) Novel alpha -D- mannosidase of rat sperm plasma membrane: characterization and potential role in sperm-egg interactions. J. Cell,. 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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35432 | - |
dc.description.abstract | 中文摘要
岩藻糖水解酶 (alpha-L-fucosidases) 為催化自糖鏈末端水解出左旋岩藻糖之酵素,其活性之喪失會造成許多重要疾病相關,包括癌症、纖維性囊腫化 (cystic fibrosis) 和岩藻糖代謝儲積症 (fucosidosis)。因此,開發具有強效及選擇性的抑制劑將有助於瞭解岩藻糖水解酶之功能和相關藥物的研究。 本實驗室先前建立一套具有fuconojirimycin結構抑制劑之快速合成篩選方法,並快速找到Thermotoga maritima及人類組織 (human tissue) 岩藻糖水解酶之強效抑制劑。值得注意的是,這些抑制劑對前者造成slow, tight-binding inhibition,然而對後者卻無此現象。這些分子因此對Thermotoga maritima岩藻糖水解酶有較佳之抑制效果,其中最強的抑制劑對兩種酵素的結合強度差異高達5400倍。 藉由序列比對及電腦模擬分析,初步推測這種截然不同的抑制情形應和Thermotoga maritima岩藻糖水解酶對於抑制劑本身aglycon的結合區有關,而人類組織岩藻糖水解酶則缺乏此一結合區。為由分子層次來研究此一現象,本論文選取6個胺基酸 (W58、F59、Y64、L191、M225、Y267) 進行定點突變分析,在突變成丙胺酸之後,slow-binding inhibition在此6個突變株均有不同程度的減弱。由蛋白質螢光實驗發現,突變型岩藻糖水解酶Y64A及Y267A在抑制劑作用下沒有明顯的結構轉變(conformational change),此點與人類岩藻糖水解酶相同。其結果不但與動力學實驗相符合,亦證實了這些疏水性胺基酸對於抑制劑algycon之結合扮演重要角色。 | zh_TW |
dc.description.abstract | Abstract
alpha-L-Fucosidases are exo-glycosidases to cleave alpha-linked L-fucose residues from the glycoconjugates that usually participate in a variety of important biological processes. Decreased α-L-fucosidase activity is related to a number of pathological conditions such as inflammation, cancer, cystic fibrosis and fucosidosis. Therefore, discovery of potent and selective inhibitors certainly sheds light on the study of their functions and development of therapeutic agents. Previously, our lab established rapid synthesis of fuconojirimycin-based inhibitors in microtiter plates for high-throughput screening, which rapidly identified potent and selective inhibitors against alpha-L-fucosidases from Thermotoga maritima and human tissue. Interestingly, slow, tight-binding inhibition was found in the study of the former enzyme, distinct from the reversible inhibition of the latter. These molecules thus have higher affinity with the Thermotoga maritima alpha-L-fucosidases than that with the human enzyme, with the best inhibitor exhibiting inhibitory difference up to 5,400-fold . According to the homology modeling and molecular docking, the drastic difference in the inhibition was probably attributed to the hydrophobic aglycon binding site that was absent in the human fucosidase. To decipher the interesting distinction at molecular level, six residues (W58, F59, Y64, L191, M225 and Y267) likely involved in the aglycon binding were changed to alanine by site-directed mutagenesis. Slow-binding inhibition was diminished to different degrees in activity assays of these mutants. Futhermore, intrinsic fluorescence analysis indicated that Y64A and Y267A mutants had no significant change upon inhibitor titration and thus behaved like their human counterpart. The result is not only consistent with the corresponding kinetic data, but also corroborates the role of proposed hydrophobic residues in the aglycon binding site. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T06:52:33Z (GMT). No. of bitstreams: 1 ntu-94-R92b46010-1.pdf: 3518032 bytes, checksum: a6b123efd2e58539add98afc946df5bf (MD5) Previous issue date: 2005 | en |
dc.description.tableofcontents | 壹. 緒論 1
一. 岩藻糖水解酶 ( alpha-L-fucosidase ) 之生物功能 1 1. 岩藻糖水解酶之簡介極其重要性 1 2. 岩藻糖水解酶之特性 2 2.1 Thermotoga maritima alpha -1,2-L-岩藻糖水解酶 2 2.2 Thermotoga maritima岩藻糖水解酶之催化反應機構 3 二. 岩藻糖水解酶抑制劑之開發 4 1. Nojirimycins—糖水解酵素抑制劑之始祖 4 2. 岩藻糖水解酶抑制劑1-Deoxyfuconojirimycin (DFJ)及其抑制機制 5 三. 加強型DFJ衍生物抑制劑之快速合成與篩選 6 四. Slow-binding inhibitor之介紹及其應用 8 1. Slow-binding inhibitor之發現 8 2. Slow-binding inhibitor之特徵及其作用機制 9 3. Slow-binding inhibitor之研究文獻回顧 11 3.1 三氯沙對醯基攜帶者蛋白還原酶之抑制研究 12 3.2 磷胺黴素對DXR之抑制研究 14 3.3 環氧化酵素抑制劑之研究 16 五. 實驗動機 20 貳. 材料與方法 21 一. 實驗材料 21 1. 藥品 21 2. 菌株及載體 21 二. 實驗儀器設備 24 三. 實驗方法 22 1. 電腦模擬計算 (Computational modeling) 22 1.1 human tissue岩藻糖水解酶結構之模擬 22 1.2 抑制劑之結合模擬 22 2. 基因選殖 (Gene cloning) 23 2.1 小量質體DNA之抽取 23 2.2 DNA瓊脂明膠電泳 24 2.3 膠體中DNA之回收與純化 24 2.4 定點突變基因之建構 24 2.5 DNA轉型作用 25 2.6 選殖基因之確認 26 3. 蛋白質純化與鑑定 (Protein purification and identification) 26 3.1 蛋白質之誘導與表現 26 3.2 蛋白質粗抽取液之取得 26 3.3 蛋白質之純化 26 3.4 蛋白質之定量 27 3.5 蛋白質聚丙醯胺膠體電泳之分析 27 3.6 蛋白質鑑定 28 4. 酵素活性分析 28 4.1 受質溶液配置 28 4.2 酵素活性之測定 29 5. 抑制劑活性分析 29 5.1 DFJ衍生物抑制劑之抑制機制測定 29 5.2 In situ screening of the inhibitor activity 30 5.3 代表性抑制劑之動力學常數測定 30 5.3.1 典型可逆型抑制劑Ki之測定 31 5.3.2 Slow-binding抑制劑之Ki及Ki*測定 32 6. 螢光光譜分析(Fluorescence spectrum analysis) 33 7. 圓二色光譜分析 (Circular dichroism analysis) 33 參. 實驗結果 35 一. T. maritima及human tissue岩藻糖水解酶序列比較 35 二. 利用同源模擬法模擬human tissue岩藻糖水解酶之結構 35 三. 抑制劑F2之molecular docking結果 36 四. 質體建構結果 36 五. 定點突變建構結果 36 六. 蛋白質表現與純化之結果 36 七. 蛋白質活性分析結果 37 1. 以p-Nitrophenyl-alpha-L-fucopyranoside為受質進行活性測定 37 2. 以4-Methylumbelliferyl- alpha -L-fucopyranoside為受質進行活性測定 37 八. 抑制劑活性分析結果 37 1. 抑制機制測定之結果 37 2. 以快速合成篩選法測試抑制劑對突變型藻糖水解酶抑制效果之結果 38 2.1 96孔盤中快速合成抑制劑之結果 38 2.2 抑制劑活性之篩選結果 38 3. 代表性抑制劑之動力學分析結果 38 3.1 化合物 1對野生株及突變株岩藻糖水解酶之抑制結果 38 3.2 化合物 2對野生株及突變株岩藻糖水解酶之抑制結果 39 3.3 化合物 3對野生株及突變株岩藻糖水解酶之抑制結果 39 九. 螢光光譜分析結果 39 十. 圓二色光譜分析結果 40 肆. 結果與討論 41 一. T. maritima及human tissue岩藻糖水解酶序列與結構模擬之比較與探討 41 二. 定點突變對酵素活性之影響 42 三. 抑制劑對野生型及突變型岩藻糖水解酶之抑制探討 42 1. 抑制機制之探討 42 2. 抑制劑結構與定點突變對slow-binding inhibition的影響 43 2.1 化合物 1之抑制探討 44 2.2 化合物 2之抑制探討 44 2.3 化合物 3之抑制探討 45 2.4 整體結果之探討 45 四. 以螢光光譜探討抑制劑結合對蛋白質結構之影響 46 五. Loop結構對受質與抑制劑結合之角色臆測 47 伍. 未來展望 49 陸. 參考文獻 50 表 圖 附錄 | |
dc.language.iso | zh-TW | |
dc.title | Fuconojirimycin C1取代衍生物對嗜熱菌岩藻糖水解酶之抑制研究 | zh_TW |
dc.title | Study of the Slow-Binding Inhibition of 1-Substituted Fuconojirimycins against Thermotoga maritima alpha-L-Fucosidase | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳新,陳佩燁,邱式鴻 | |
dc.subject.keyword | 岩藻糖水解酶,抑制劑, | zh_TW |
dc.subject.keyword | slow-binding,fucosidase,inhibitor,fuconojirimycin, | en |
dc.relation.page | 93 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2005-07-28 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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