中文摘要
植物液泡(Vacuole)在植物細胞中主要功能為細胞質恆定之維持及儲存生理代謝物質。植物液泡執行其功能時主要藉由液泡膜系上各種通道或輸送性蛋白來完成，其中有兩種磷酸水解酵素:液泡H+-ATP水解酵素(vacuolar H+-ATPase)和液泡H+-PPase水解酵素(Vacuolar H+-PPase)為驅動這些液泡之通道或輸送性蛋白的能量轉換裝置，因為其能量來源主要由ATP和PPi提供。目前植物液泡H+-ATP水解酵素(H+-ATPase)之研究在其功能和生理角色上已經有相當豐富的成果和了解。但液泡H+-PPase水解酵素(H+-PPase)至目前仍有很大研究了解的空間。本研究就從化學修飾抑制及基因定點突變的角度，來了解液泡H+-PPase水解酵素之化學修飾抑制作用之成因，藉以標定酵素中功能性胺基酸的位置和角色。並可提供研究膜蛋白質或酵素之結構與功能之概念和方法。
首先以組胺酸(Histidine)之專一化學修飾劑diethylpyrocarbonate
(DEPC)對綠豆液泡H+-PPase進行化學修飾抑制研究，顯示DEPC可以抑制綠豆液泡H+-PPase之PPi水解酵素活性及質子輸送功能。且綠豆液泡H+-PPase之受質Mg2+-PPi 可保護綠豆液泡H+-PPase之酵素活性中心不被DEPC之化學修飾抑制，顯示可能有DEPC修飾之組胺酸為綠豆液泡H+-PPase之功能上重要胺基酸基團。另外，已知綠豆液泡Cys-630為酵素活性中心鄰近的胺基酸基團，已被證實Cys-630可被其專一性化學修飾劑NEM修飾但不會抑制酵素活性，利用此修飾劑進行先期化學修飾再利用DEPC對綠豆液泡H+-PPase進行抑制動力學研究，顯示DEPC對綠豆液泡H+-PPase抑制值由1.1histidine/subunit下降至0.7 histidine /subunit，顯然NEM修飾作用可以減緩DEPC對綠豆液泡H+-PPase抑制作用。綜合上述結果，我們認為可能有一個DEPC修飾之組胺酸鄰近或位於酵素活性中心。
基於以上結果，我們要進一步確認DEPC修飾之標的組胺酸位置，而利用基因定點突變，和酵母菌異體基因表現與微小體(Microsome)分離，且從酵母菌基因體序列中並未比對出可能為H+-PPase存在酵母菌基因體中，經西方點墨(Western bolt)，PPi水解活性鑑定及質子輸送之測定結果顯示液泡H+-PPase是可以大量被遞送(Targeting)到酵母菌內膜系統中且具有液泡H+-PPase之活性，故我們基於這些基本條件進行異體基因表現研究。經由胺基酸序列比對發現有6個組胺酸存在綠豆液泡H+-PPase中，其中H716在不同生物之H+-PPase胺基酸序列中為唯一具有高度保留性之組胺酸，經基因定點突變和酵母菌異體基因表現，當H716被置換成H716A之定點突變株會降低PPi水解活性和質子輸送功能。經由計算其PPi水解/質子輸送效率顯示比正常株下降約50%，再者H716A突變也會改變液泡H+-PPase之PPi最佳水解環境之pH值，有0.8單位的酸性偏移現象，顯示Histidine被置換成Alanine後液泡H+-PPase之PPi水解活性和質子輸送功能會受影響。已知F-可藉由形成Mg2+-F-2複合物會干擾液泡H+-PPase之PPi水解活性，實驗結果顯示H716A突變會降低F-抑制PPi水解活性作用達正常株之1/2。由熱處理T1/2值之評估得知各種Histidine置換成Alanine之突變株並未看出有明顯改變H+-PPase之熱穩定性，H716A亦然。如將H716A之突變株再用DEPC進行化學修飾抑制，顯示DEPC抑制性只有相對於正常株之1/3，而其他突變株未顯示相當之耐受性，亦即DEPC修飾主要對象是H716。另外，利用Trypsin 進行部分水解(limited digestion)研究顯示Mg2+-PPi未能在H716A突變株上呈現與正常株一樣的受質保護現象，顯示H716A有可能引發酵素活性中心結構改變以致使酵素受質保護現象消失，但也不排除因結構改變而間接影響受質保護現象。以上結果顯示H716可能參與液泡H+-PPase之PPi水解活性及質子輸送功能或是酵素活性之調節功能上，且H716也是DEPC修飾抑制液泡H+-PPase活性主要成因。
其次，前人研究結果顯示利用精胺酸(Arginine)專一性化學修飾劑phenylglyoxal(PGO)及2,3-Butanedione(BD)進行化學修飾，能抑制液泡H+-PPase之PPi水解活性及質子輸送功能，由抑制動力學研究顯示可能有一個或一個以上之化學修飾之精胺酸為液泡H+-PPase之功能上必要之基團，此基團亦會因Mg2+-PPi保護酵素活性中心而降低化學修飾抑制作用。因此，引發我們有興趣要鑑定出此化學修飾之標的，首先由胺基酸序列比對發現共有15個精胺酸存在綠豆液泡H+-PPase中，再進行基因定點突變及酵母菌異體基因表現和微小體分離獲得各種精胺酸定點突變之液泡H+-PPase進行分析。目前已標的出R242很可能為此化學修飾之標的，因為R242A突變使液泡H+-PPase之PPi水解活性及質子輸送功能喪失95%之活性，在最佳PPi水解環境之pH值之觀察，R242A突變會引發最佳PPi水解環境pH值有1.0單位之酸性偏移的現象，進一步觀察精胺酸專一性化學修飾劑PGO及BD之修飾抑制顯然對R242A突變株並未產生抑制作用，在高濃度之F-存在下觀察PPi水解活性作用，F-抑制R242A突變株只有正常之株之1/6，綜合上述結果，我們相信R242應該是精胺酸化學修飾劑抑制之主要對象，但也不能排除因失去電價性側鏈引發結構改變之間接影響。總之，我們結合化學修飾抑制及基因定點突變成功的標的出功能性胺基酸之位置及其角色如何，也提供蛋白質結構功能研究上，另一種研究方法及概念。

Abstract

Vacuolar H+-pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at expense of PPi hydrolysis. The identification of gene encoding an amino acid sequence demonstrates that vacuolar H+-PPase of mung bean contains 6 histidine residues and 15 arginine residues. This study showed vacuolar H+-PPase may contain several histidine and arginine residue(s) essential for the enzymatic activity and H+-translocation of vacuolar H+-PPase. Furthermore, we identified the roles of histidine and arginine residues in mung bean vacuolar H+-PPase by site-directed mutagenesis. A line of mutants with histidine and arginine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Among histidine mutants scrutinized, only did the mutation of H716 decrease the enzymatic activity, the proton transport and the coupling ratio of vacuolar H+-PPase. The mutation at H716 of vacuolar H+-PPase shifted the optimum pH value but not the T1/2 (pretreatment temperature at which half enzymatic activity is observed) for PPi hydrolytic activity. Mutation of H716 is obviously declined the effect of substrate protection on the vacuolar H+-PPase as determined by immunoblotting analysis after limited trypsin digestion. Moreover, mutation of these histidine residues modified the inhibitory effects of F- and Na+, but not that of Ca2+. Single substitution of H704, H716 and H758 by alanine released the effect of K+ stimulation, indicating possible location of K+ binding in the vicinity of domains surrounding these residues. A working topological model is thus proposed to elucidate the roles of crucial histidines.
As for arginine mutated variants, R242A, R523A, and R609A mutants displayed declined activity of PPi hydrolysis and proton translocation than the wild-type. These mutants showed a shift in optimal pH for enzymatic activity. Among arginine mutants, R242A is relatively resistant to PGO and BD treatments, suggesting that it is the primary target for the attack of these modifiers. Furthermore, only did R242A mutant displayed relatively lower sensitivity to F- inhibition under similar conditions. Taken together, we speculate that R242 may locate in active domain of vacuolar H+-PPase. A working model is proposed to accommodate information from previous studies on the catalytic site of vacuolar H+-PPase.

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