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研究生:邱性嘉
研究生(外文):Xing-Jia Qiu
論文名稱:豬腎之N-acetyl-D-glucosamine 2-epimerase ATP結合位的探討
論文名稱(外文):Characterization of ATP-binding site in porcine N-acetyl-D-glucosamine 2-epimerase
指導教授:李晏忠
指導教授(外文):Yen-Chung Lee
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物農業科技學系研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
畢業學年度:103
語文別:中文
論文頁數:50
中文關鍵詞:N-acetyl-D-glucosamine 2-epimeraseN 端定序LC-MS/MSATP-footprintingATP結合位
外文關鍵詞:N-acetyl-D-glucosamine 2-epimeraseN-terminal sequencingLC-MS/MSATP-footprintingATP-binding site
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N-acetyl-D-neuraminic acid (NeuAc) 為 sialic acid 中主要的化合物,在許多的生物辨識過程中扮演重要的角色。NeuAc 之衍生物已被作為各種臨床用藥,如用於抑制流感病毒的 neuraminidase 的藥物。N-acetyl-D-glucosamine 2-epimerase (AGE) 能夠催化 N-acetyl-D-glucosamine (GlcNAc) 與 N-acetyl-D-mannosamine (ManNAc) 之間可逆的表異構化反應,而 ManNAc 即為 NeuAc 之前驅物。 AGE分別具有一個受質結合的催化部位 (catalytic site) 與一個 ATP 結合的調節部位 (allosteric site)。來自於 Anabaena sp. CH1 之 AGE (bAGE) 的 ATP 結合位已藉由 ATP-footprinting的方式證明是位於 156-GKYTK-160 的 flexible loop 區域上,然而,關於豬腎 AGE (pAGE) 的ATP 結合位置則尚未清楚。本研究中因此也利用 ATP-footprinting 的方式來探討 pAGE 確切的 ATP 結合部位。在 ATP 存在下,pAGE 會對 trypsin 的水解具有抗性,相對的,在缺少 ATP 的情況下,trypsin會將 pAGE 水解為 26 kDa 及 20 kDa 兩個片段。蛋白質 N 端定序結果顯示兩個片段分別為 pAGE 的 N 端及 C 端片段。LC-MS/MS 分析結果顯示在缺少 ATP 的情況下,trypsin 會專一性的辨識 R220 殘基,並將 pAGE 水解為兩個片段。定點突變與分析發現即使在 ATP 存在下,pAGE 的突變蛋白 R212A、R213A 及 R220A 完全失去 ATP-footprinting 的特性及酵素活性。相反的,R167A 顯示與野生型 pAGE 有相似的 ATP-footprinting 特性及酵素活性。R159A 則具有些微的 ATP footprinting 的現象及 28 % 的相對活性。本研究結果顯示 pAGE 之 R212、R213 及 R220 殘基在 ATP 的結合上扮演重要的角色。不同於 bAGE 的 ATP binding loop,本研究首度證實了 pAGE 之 ATP binding site 是位於 R220 殘基所在的 loop,即位於 α-Helix 8 及 α-Helix 9 之間 R212–C239 的區域。
N-acetyl-D-neuraminic acid (NeuAc), a major species of the sialic acids, plays important roles in various biological recognition processes. NeuAc derivative analogs have been used as clinical anti-influenza drugs to inhibit neuraminidase of influenza virus. N-acetyl-D-glucosamine 2-epimerase (AGE) catalyzes the reversible epimerization between N-acetyl-D-glucosamine and N-acetyl-D-mannosamine that serves as the precursor of NeuAc. AGE possesses two distinct interaction sites, a catalytic site for substrate binding and an allosteric site for ATP binding. The ATP binding site of Anabaena sp. CH1 AGE (bAGE) has been verified by ATP-footprinting, and the result revealed that it was located at a flexible loop region of 156-GKYTK-160. However, the counterpart ATP binding site of pocine AGE (pAGE) has not yet been clearly identified. In this study, we intend to verify the ATP binding site of pAGE through the ATP-footprinting strategy. In the presence of ATP, pAGE was resistant to trypsin digestion. Contrariously, trypsin digested pAGE into 26 kDa and 20 kDa segments in the absence of ATP. N-terminal sequencing analysis revealed that 26 kDa and 20 kDa were belong N-terminal and C-terminal portion of pAGE, respectively. LC-MS/MS analysis demonstrates that trypsin specific recognized R220 residue and digested pAGE into 2 segments in the absence of ATP. In the presence of ATP, the pAGE variants R212A, R213A and R220A completely loss of ATP footprinting properties and enzymatic activities. In the contrary, the R167A variant showed ATP footprinting pattern and enzyme activity similar with wild type pAGE. The R159A variant also has slight similar pattern and remain 28 % of relative activity. These results demonstrate that the R212, R213 and R220 residues of pAGE may play important roles in ATP binding. The R220 residue located in a flexible loop ranged α-Helix 8 between α-Helix 9 which is different from the ATP binding loop found in bAGE. This is the first report that the ATP-binding site of pAGE locate in the region of R212–239.
中文摘要........................................i
英文摘要........................................ii
致謝...........................................iii
前言............................................1
一、N-acetyl-D-glucosamine 2-epimerase (AGE)....1
1.1 AGE 與 renin-binding protein (RnBP).........1
1.2 核苷酸對 AGEs 之作用..........................2
1.3 AGE 之結構...................................3
二、AGE 之 ATP 結合位.............................4
三、研究目的......................................4
材料與方法........................................5
一、藥品..........................................5
二、菌株、質體及培養基..............................5
三、DNA 之操作....................................5
四、pAGE 之定點突變................................5
五、重組蛋白 pAGE 之表現與純化.......................6
六、蛋白質電泳分析 SDS-PAGE.........................7
6.1 SDS-PAGE 膠體的製備...........................7
6.2 SDS-PAGE 分析................................7
七、蛋白質定量.....................................7
八、pAGE 之 ATP-footprinting 作用分析..............8
九、蛋白質 N 端定序................................8
十、LC-MS/MS 分析.................................8
十一、酵素活性分析..................................9
11.1 GlcNAc 及 Neu5Ac 標準曲線之建立...............9
11.2 pAGE 之活性分析..............................9
結果.............................................10
一、重組蛋白 pAGE 之表現與純化.......................10
二、pAGE 之 ATP-footprinting 作用分析..............10
三、pAGE 之 ATP 結合位分析.........................11
四、pAGE 之酵素活性分析............................11
4.1 GlcNAc 及 ManNAc 標準曲線之建立................11
4.2 pAGE 之活性分析...............................12
五、pAGE 結合 ATP 之分子模擬 (ATP docking)..........13
討論.............................................14
參考文獻..........................................17
圖表..............................................20
附錄..............................................37
附錄一、pAGE 之基因與蛋白質序列.......................37
附錄二、培養基配方..................................38
附錄三、Favorgen 套組操作流程........................39
附錄四、定點突變之流程...............................40
附錄五、實驗相關溶液之配方............................41
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