離子泵焦磷酸水解酶(M-PPase)此酵素主要存在於植物、細菌、以及一些人類的致病菌中，主要藉由水解焦磷酸所產生的能量來驅使細胞膜或液胞膜外質子或鈉離子的轉運，M-PPase對這些物種生存於惡劣環境的耐受性上扮演一個關鍵的角色。至今為止，只有綠豆及海棲熱袍菌M-PPase的分子結構被解析出來，M-PPase的蛋白質序列具有高度的相似性，並且雙磷酸鹽具有專一性抑制M-PPase水解的能力，因此我們認為M-PPase是個具有潛力的藥物設計目標，希望以VrH+-PPase的結構為藥物設計的模板並以結晶的方法觀察VrH+-PPase與雙磷酸鹽的交互作用。
　　雙磷酸鹽是一種常見治療骨質疏鬆的藥物，其主要的作用機制是藉由磷-碳-磷(P-C-P)鍵結來取代三磷酸腺苷(ATP)或焦磷酸(PPi)的磷-氧-磷(P-O-P)鍵結，進而抑制三磷酸腺苷水解酶的活性，如此一來，生物體產生的能量將會減少，使得破骨細胞漸漸的凋亡，以達到治療骨質疏鬆的效果，因此希望以舊藥新用的概念將雙磷酸鹽用於治療帶有M-PPase的致病菌所造成的疾病。
　　本研究中，我們解析了VrH+-PPase與1-羥基-亞乙基-1,1-二膦酸(Etidronate；EA)、3-氨基-1-羥基-亞丙基-1,1-二膦酸(Pamidronate；PA)、4-氨基-1-羥基-亞丁基-1,1-二膦酸(Alendronate；AA)等三種雙磷酸鹽分子的複合體結構。由我們實驗室先前研究VrH+-PPase-IDP的複合體結構中VrH+-PPase擁有五個Mg2+協助IDP的結合，其中，Mg2和Mg3是與酵素活性相關的輔因子，Mg1和Mg4會與PPi形成Mg2PPi複合物，在VrH+-PPase-EA複合體結構中，EA側鏈的氧原子可以跟酵素的Mg3交互作用，而PA及AA側鏈的氮原子取代了基質的Mg1的功能，此結果顯示了不同的雙磷酸鹽在VrH+-PPase的結合位中會產生不同的方向性及交互作用力。此外，在水解焦磷酸活性抑制的實驗中可以發現，抑制效果的順序為，亞氨二磷酸(Imidodiphosphate；IDP)＞EA＞PA＞AA，VrH+-PPase與IDP的結構中，具有較多的Mg2+ (5個Mg2+)來幫助彼此間緊密的交互作用，而且其化學結構與焦磷酸最為相似，或許可以更穩定的結合VrH+-PPase。

　　Membrane-embedded pyrophosphatases (M-PPases) are found in plants, bacteria, and several human pathogens. These enzymes hydrolyze pyrophosphate and trigger the translocation of ions (H+ and/or Na+) across plasma/vacuolar membranes. M-PPases play a key role in survivability and stress tolerance. So the M-PPases are the potential target protein in the drug-design. Currently, only the structures of VrH+-PPase from Vigna radiata and TmNa+-PPase from Thermotoga maritima are solved. The protein sequences of M-PPases are highly conserved, and bisphosphonates are the type-specific inhibitors of M-PPases. Thus, M-PPases could be the potential target in the drug-design, and the structure of VrH+-PPase is utilized as a model for drug-design.
　　Bisphosphonates are the common drugs to treat Osteoporosis. In the chemical structure, bisphosphonates are composed of P-C-P backbone which is similar to pyrophosphate or adenosine triphosphate (ATP). In addition, bisphosphonates competitively inhibit the hydrolytic activity of ATPases and M-PPases, which result in the reduction of energy production from ATPases. The osteoclast will then gradually undergo apoptosis to achieve the treatment of Osteoporosis. Take the concept of drug repurposing, bisphosphonates may be used to treat the diseases caused by M-PPase containing pathogens since bisphosphonates inhibit M-PPases on energy production.
　　In this study, we solved the structures of VrH+-PPase with three various bisphosphonates, including Etidronate (EA), Pamidronate (PA) and Alendronate (AA). Comparing to the previous VrH+-PPase and imidodiphosphate (IDP) complex structure, the oxygen of EA side chain interacts with enzyme-type Mg3, and the nitrogen of PA and AA side chain occupies the substrate-type Mg1. It suggests that the orientations of those bisphosphonates in the VrH+-PPase binding pocket are different from IDP. In the study of hydrolytic inhibition, IDP has the best inhibitory ability and AA is the worst one. Combining the structural and inhibitory studies, we can conjecture VrH+-PPase has the most Mg2+ to interact with IDP and IDP is the most similar to pyrophosphate, so IDP can tightly interact with VrH+-PPase.

中文摘要 I
Abstract II
致謝 III
目錄 IV
第一章 1
簡介 1
1.1 離子泵酵素 (Ion-pumping enzyme) 1
1.2 膜鑲嵌型焦磷酸水解酶 (Membrane-embedded pyrophosphatase；M-PPase) 2
1.3 綠豆的質子泵焦磷酸水解酶 (VrH+-pyrophosphatase；VrH+-PPase) 3
1.4 雙磷酸鹽 (Bisphosphonate) 3
第二章 6
實驗方法與材料 6
2.1 酵母菌表現系統表現VrH+-PPase 6
2.2 微粒體的分離及蛋白的純化 7
2.3 製備酵母菌的液胞膜 8
2.4 點突變 9
2.5 蛋白質定量 10
2.6 SDS-PAGE和Western blotting 10
2.7 焦磷酸水解活性 11
2.8 質子轉運實驗 12
2.9 VrH+-PPase的結晶實驗 13
第三章 14
結果與討論 14
3.1 M-PPase蛋白質序列比對 14
3.2 VrH+-PPase質子轉運的能力 14
3.3 雙磷酸鹽抑制VrH+-PPase水解PPi的能力 15
3.4 VrH+-PPase-EA的複合體結構 16
3.5 VrH+-PPase-PA的複合體結構 17
3.6 VrH+-PPase-AA的複合體結構 18
3.7 比較VrH+-PPase-IDP,-EA,-PA,-AA複合體 18
3.8 VrH+-PPase的突變 19
第四章 22
結論 22
第五章 23
表格與圖片 23
第六章 46
參考文獻 46

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