膜蛋白焦磷酸水解酶(M-PPases) (EC 3.6.1.1)，藉由水解焦磷酸產生能量驅動質子或鈉離子進入細胞胞器。此酵素存在於植物、細菌、以及原生生物。先前的研究提出焦磷酸的類似物雙磷酸鹽具有抑制M-PPases水解焦磷酸的能力，例如亞氨基二磷酸（imidodiphosphate；IDP）和亞甲基二磷酸鹽（methylenediphosphonate；MDP）等。本實驗室於2012年成功解析出Vigna radiata H+-PPase(VrH+-PPase)及IDP複合物的三度空間的晶體結構。此次研究主題是以X光晶體繞射的方法決定VrH+-PPase與MDP複合物的分子結構，並探討MDP抑制VrH+-PPase的水解能力。VrH+-PPase-MDP複合物晶體結構解析度為3.1Å，其MDP結合區域由13個酸性胺基酸、3個離胺酸和1個天門冬胺酸，且包含1個MDP分子、5個鎂離子(Mg1-5)和1個鉀離子。我們也觀察到MDP抑制VrH+-PPase的水解能力較IDP弱，其抑制常數(Kappi)為838.8 μM。VrH+-PPase-MDP與VrH+-PPase-IDP複合物分子結構類似， VrH+-PPase-MDP的複合物中Mg5與D530產生新的交互作用力，此外親核基水分子(WATnu (nucleophilic water)位置不一樣。MDP抑制PPi水解效果低於IDP，推測因為MDP的碳原子無電子對因而無法與Mg5產生交互作用力，而MDP於單元體A中只與K694、K730產生氫鍵，於單元體B中反而與K250和K694形成氫鍵。而且MDP與鎂離子之間距離較遠長，兩者之間作用力降低，因此MDP與VrH+-PPase結合不如IDP來得緊密紮實，IDP仍是目前最強的抑制劑。

Membrane-bound pyrophosphatases (M-PPases) (EC 3.6.1.1), couple the PPi hydrolysis to pumping of monovalent cations across cellular membrane into the organelle. Most of the M-PPases exist in higher plants, bacteria, and some human pathogens. Previous studies have suggested that pyrophosphate analogs inhibit the ability of H+-PPases to hydrolyze, such as imidodiphophate (IDP) and, methylenediphosphonate (MDP). Our laboratory successfully solved the crystal structure of Vigna radiata H+-translocating pyrophosphatase with IDP complex (VrH+-PPase-IDP complex) in 2012. We determined the crystal structure of VrH+-PPase-MDP complex by crystallography and X-ray diffraction, the MDP binding pocket contains 13 acidic residues、three lysine residues and one asparagine residue. In addition, there are five magnesium ions and one potassium ion around MDP. Also we investigated the ability of MDP to inhibit the pyrophosphate hydrolysis of VrH+-PPase and obtain its inhibition constant Kappi ) as 838.8 μM in this study. The overall structure of the complex of VrH+-PPase-MDP is similar to that of the VrH+-PPase-IDP complex. In VrH+-PPase-MDP complex, there is additional interaction between Mg5 and D530. The positions of nucleophilic water (Watnu) for PPi hydrolysis between MDP and IDP complexes are varied. The inhibition effect of MDP for PPi hydrolysis is weaker than that of IDP. Because the C atom of P-C-P in MDP hasn’t that lone pair dlectron to interact with Mg5. MDP formed hydrogen bonds with K694 and K730 in monomer A. But in monomer B, MDP. Because of the degree of P-C-P in MDP is smaller than P-N-P in IDP, the bonding distances between Mgs and MDP are longer in the VrH+-PPase-MDP complex, and the interaction are decreased. Up to now, IDP is still the strongest inh ibitor for PPi in M-PPase.

中文摘要 .................................................................................................................................... I
Abstract .................................................................................................................................... II
誌謝 .......................................................................................................................................... III 第一章 簡介 ............................................................................................................................. 1 1.1焦磷酸水解酶分類 ......................................................................................................... 1 1.2膜鑲嵌型焦磷酸水解酶 (Membrane-embedded pyrophosphatase；M-PPase) ...... 1 1.3質子泵焦磷酸水解酶之生理意義 ................................................................................. 2 1.4綠豆質子泵焦磷酸水解酶(VrH+-PPase)結構 .......................................................... 3
1.5雙磷酸鹽類 .............................................................................................................. 4
1.6 IDP與MDP之分子結構與功能 ............................................................................. 5
第二章 實驗材料與方法 ................................................................................................... 6
2.1 轉殖酵母菌表現焦磷酸水解酶VrH+-PPase (transformation) ................................. 6
2.2 放大與誘導酵母菌表現膜蛋白 VrH+-PPase .............................................................. 6
2.3 溶解酵母菌的細胞壁 .................................................................................................... 7
2.4製備微粒體 ..................................................................................................................... 7
2.5純化膜蛋白 VrH+-PPase ............................................................................................... 8
2.6 VrH+-PPase-MDP 複合物晶體生成環境 .................................................................... 9
2.7 VrH+-PPase-MDP複合物X-ray 繞射點數據收集，及分子置換法之結構判定 ... 10
2.8 Bradford蛋白質定量法 .............................................................................................. 11
2.9 十二烷基硫酸鈉聚丙烯酰胺凝膠電泳(SDS-PAGE) ................................................ 12
2.10製備含有VrH+-PPase的液泡膜 ............................................................................... 12
2.11水解活性分析 ............................................................................................................. 14
第三章 結果 ........................................................................................................................... 15
3.1 VrH+-PPase-MDP 複合物晶體之培養 ...................................................................... 15
3.2 VrH+-PPase-MDP 複合物晶體之空間群(space group) ........................................... 15
3.3 VrH+-PPase-MDP 複合物晶體結構 .......................................................................... 16
V
3.4 MDP分子抑制VrH+-PPase焦磷酸水解結果 ........................................................... 17
第四章 討論 ........................................................................................................................... 19
4.1 五種雙磷酸抑制VrH+-PPase水解焦磷酸之效果 .................................................... 19
4.2 VrH+-PPase-MDP 複合物之單元體 A和B 之MDP結合區域之差異 ................ 21
4.3 MDP結合區域胺基酸、鎂離子和MDP分子三者之間關係 .................................. 22
4.4 MDP結合區域之離胺酸(lysine；K250、K694、K730) 與MDP分子產生氫鍵 24
4.5 VrH+-PPase-MDP 複合物之單元體 A和B 之Watnu之差異處 ......................... 25
第五章 表格與圖 ................................................................................................................... 27
第六章 參考文獻 ................................................................................................................... 38

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