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研究生:潘羿娟
研究生(外文):Yih-Jiuan Pan
論文名稱:丙氨酸掃描式突變分析綠豆液泡焦磷酸水解酶之第六穿膜區
論文名稱(外文):Alanine-scanning Mutagenesis along Transmembrane Domain Ⅵ of the Mung Bean Vacuolar H+-Pyrophosphatase
指導教授:潘榮隆潘榮隆引用關係
指導教授(外文):Rong-Long Pan
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:45
中文關鍵詞:焦磷酸水解酶穿膜區丙氨酸掃描式突變分析螺旋環柱偶合效應質子傳送
外文關鍵詞:pyrophosphatasetransmembrane domainalanine-scanning mutagenesishelix wheelcoupling efficiencyproton translocation
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已知植物液泡膜上焦磷酸水解酶(簡稱V-PPase)在維持細胞質的中性酸鹼度扮演很重要的角色:V-PPase 藉由水解焦磷酸驅動質子由細胞質輸入液泡中,以維持細胞質的中性酸鹼度。由於對於焦磷酸水解酶的結構尚未清楚,因此根據TopPred Ⅱ軟體所預測的地誌模式,受質水解的催化位置被認為是位在第五與第六穿膜區之間的環區且面向在細胞質中。目前吾人還不清楚是否此催化區會與相鄰的穿膜區互相影響,或是與穿膜區上個別的胺基酸有相互關係。第六穿膜區在不同物種間有高度相似性,並且可能參與催化功能或維持結構的穩定性。在此篇論文,我們利用點突變的方法,分別將21個胺基酸置換成丙氨酸,在酵母菌內異體表現綠豆的焦磷酸水解酶,並測其活性與質子傳送作用以觀察第六穿膜區的重要性。結果發現Y299A, E301A,A306S, L307A, L317A 與N318A突變株不論水解活性或是偶合效率均嚴重受損。這些突變株在第六穿膜區的位置恰巧分布在兩端與中間,且其在整各螺旋環柱的分佈均分三區。由此猜測,這三區具有維持穩定結構的功能。A305S, V308A, A310S, G316A與H319A仍保有活性但降低了偶合效率,且位在螺旋環柱的同一面,暗示這些胺基酸可能與質子運送有關。另外,藉由離子效應的研究,我們也發現Y299A, C304A, L307A, V308A, L317A與N318A突變株對於鉀離子、鈣離子、鈉離子與氟離子均不敏感,暗示可能參與這些離子的結合。而E301突變株在離子效應除了氟離子以外也有同樣的情形。在酵素酶切實驗中,A305S, A306S, L307A, 與N318A突變株較能抵抗酵素的酶切反應;其中除了A305S 以外的突變株水解活性都很低。因此,我們認為這些突變株的低水解活性是由於其蛋白構型改變所導致。
The vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1) plays a significant role in the maintenance of pH value of cytoplasm via the proton translocation from cytosol to vacuolar lumen at the expense of PPi hydrolysis. The overall structure of the membranes-associated PPases is unclear. According to the topology of V-PPase prediced by TopPred Ⅱ, the motif of catalytic site for substrate hydrolysis was presumably located in loop V and exposed to the cytosol. It was not certain whether the motif of catalytic site interacts with adjacent transmembrane domains (TM), nor is the detailed correlation between the different amino acids. TM VI is relatively conserved and believed to participate in catalytic function and conformation stability. In this study, we carried out alanine scanning mutagenesis along TM VI of the mung bean V-PPase. Different amino acids were substituted singly by alanine and a series of site-direct mutants were constructed, over-expressed in Saccharomyces cerevisiae, and their enzymatic activities and proton translocations were determined. Y299A, E301A, A306S, L307A, L317A and N318A mutants exhibited gross impairment of both PPi hydrolysis and coupling efficiencies. The deterioration of these reactions at both ends and the middle of TM Ⅵ and the location of these amino acids at trisection of the helix wheel suggest their roles in maintaining the stability of conformation. A305S, V308A, A310S, G316A, and H319A exhibited an injury of proton translocation ability and show a decrease in the coupling efficiency but not PPi hydrolysis. They also locate at one face of the helix wheel, implicating their involvement in proton translocation. Studies on ion effects, it showed that the Y299A, C304A, L307A, V308A, L317A and N318A mutants are less sensitive to the K+, Ca2+, Na+, and F-, suggesting they exert effects on binding of these ions. Further evidence indicates that E301 may be involved in binding of these ions but not F-. From the proteolysis analysis, the A305S, A306S, L307A, and N318A mutants were relatively resistant the proteolysis. Moreover, their PPi hydrolysis activities were significantly inhibited expect A305S mutant. We suggest the low enzymatic activity of these mutants is due to the conformational change.
Introduction----------------------------------------------1
Materials and Methods-------------------------------------6
Results--------------------------------------------------15
Discussion-----------------------------------------------22
References-----------------------------------------------29
Figures and Tables---------------------------------------33
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