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研究生:李保蓁
論文名稱:植物液胞膜上焦磷酸水解酵素組胺酸716之突變分析
論文名稱(外文):Mutagenic Analysis of Histidine-716 of 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:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:44
中文關鍵詞:植物液胞焦磷酸水解酵素定點突變
外文關鍵詞:V-PPasesite-directed mutagenesisvacuole
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我們已知植物液胞膜上焦磷酸水解酵素(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打入液胞中,以維持細胞質的中性酸鹼度。在以diethylpyrocarbonate修飾綠豆V-PPase的研究中,我們已經知道組胺酸可能有參與催化水解的過程(Hsiao, Y. Y., Van, R. C., Hung, H. H., and Pan, R. L. (2002) Journal of Protein Chemistry 21: 51-58)。在對6個V-PPase組胺酸的突變分析中顯示,組胺酸716可能是參與催化過程的胺基酸,在此篇論文,我們利用點突變的方法,將組胺酸716以其他12個胺基酸取代之,並測其活性。結果發現,所有的突變株,不論是焦磷酸的水解活性,或是質子的運送能力均下降﹔H716W與H716F突變株不但仍保有被鉀離子激發的特性,而且還保有60%以上的焦磷酸水解活性及28%以上的質子傳送能力,較保留突變株(H716K與H716R)或相反特性突變株所保有的活性,均高出許多,由此推測,芳香環在這個位置是重要的;突變株及正常的焦磷酸水解酵素的最適水解活性均在pH 7.5,且在熱穩定的實驗中,均在53℃時活性急速下降,表示在716突變不會造成水解區域的結構改變﹔在外加imidazole的實驗中,所有突變株活性的結果非常分歧,推翻組胺酸716是直接參與催化過程的假設。綜合所有實驗的結果,我們推測組胺酸716在V-PPase的C端,會間接的影響焦磷酸水解、質子傳輸、及被鉀離子激發的活性。

Plant vacuolar H+-translocation inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) has been considered to play a significant role on the maintenance of pH value of cytoplasm via the proton translocation from cytosol to vacuolar lumen as being triggered at the expense of PPi hydrolysis. The diethylpyrocarbonate (DEPC)-modification study of Vigna radiata V-PPase has demonstrated the involvement of histidyl residues in or near the catalytic site of the enzyme (Hsiao, Y. Y., Van, R. C., Hung, H. H. and Pan, R. L. (2002) Journal of Protein Chemistry 21: 51-58). The mutagenic analysis of six histidines revealed that His-716 might be the candidate participating in catalytic process. His-716 was substituted with 12 different amino acids and examined the enzymatic properties. All mutants exhibited the diminution of PPi hydrolytic and H+ translocation activities. Only did H716W and H716F V-PPase hold the potassium-stimulated properties and retain more than 60 % of PPi hydrolytic and 28 % of proton transport activity. The conserved (H716K and H716R) or converse (H716D) mutations all lost their enzymatic activities, suggesting the importance of aromatic ring in this position. The optimum PPi hydrolytic activity of V-PPase of wild-type and mutants were all at pH 7.5. Moreover, the PPi hydrolytic activity of all proteins dramatically declined at around 53℃. Therefore, the mutation on His-716 did not affect the conformation of hydrolytic pocket. Imidazole preincubation was used to clarify the role of His-716 in PPi hydrolysis. The distinct outcomes of all mutants after the addition of exogenous imidazole refute the notion that His-716 directly participated in the enzymatic domain. Instead, His-716 plays roles in the long distant or indirect effects on PPi hydrolytic, H+ transport, and K+-sensitive activities on the C-terminus of V-PPase.

Introduction---------------------------------------------1
Materials and Methods------------------------------------5
Results-------------------------------------------------14
Discussion--------------------------------------------- 18
References----------------------------------------------25
Tables and Figures--------------------------------------30

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