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標題: | 啤酒花腺苷酸異戊二烯基轉移酵素對多磷酸雙腺苷之結合與催化活性之探討 Investigation on Binding and Catalytic Activity of Adenylate Isopentenyltransferase from Humulus lupulus to Diadenosine Polyphosphates |
作者: | Feng-Yuan Chen 陳鋒源 |
指導教授: | 王惠鈞 |
關鍵字: | 多磷酸雙腺苷,磷酸雙腺苷,在植物中的存在,啤酒花腺苷,酸異,戊二烯基轉移酵素,結合親和力,催化活性,點突變, ApnA,the existence of ApnA in plant,HlAIPT,binding affinity,specific activity,point mutation, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 多磷酸雙腺苷(diadenosine polyphosphates, ApnA)在生物體內是一種非常重要的訊息分子。在細胞內,它們被視為是牽涉細胞對環境壓力產生反應、去氧核糖核酸(DNA)的複製與修復、以及細胞凋零的胞內訊息分子。此外,在細胞外,它們在心血管系統中會刺激血管壁平滑肌細胞的增生、亦影響血管脈管壓力;在神經內分泌系統內,其亦作為一種神經傳導物質。其重要性是不言可喻。雖然多磷酸雙腺苷廣泛存在於各種細菌、真菌、及動物體內,植物體內是不是存在著這類物質迄今卻尚未被證實。然而,在植物體確實存在著合成以及分解這類分子的酵素,這件事間接地證實了多磷酸雙腺苷在植物中的存在。而儘管多磷酸雙腺苷在植物中調控的生物標的目前仍不清楚,多磷酸雙腺苷在植物中的量似乎被一系列的酵素微妙地調控著。利用等溫滴定微量熱法測量不同多磷酸雙腺苷與啤酒花腺苷酸異戊二烯基轉移酵素的結合能力的實驗結果顯示,不同的多磷酸雙腺苷對酵素具有不同的結合親和力,而且其中的五磷酸雙腺苷對酵素的結合親和力竟然出乎意料地高於原來已知的受質(三磷酸腺苷)。其結合親和力的關係如下:Ap5A > ATP > Ap4A > Ap6A > Ap3A。進一步測量這些多磷酸雙腺苷對酵素的活性並和酵素對三磷酸腺苷的活性作比較後,其活性大小的關係為ATP > Ap6A > Ap4A > Ap5A > Ap3A。這些實驗結果意味著多磷酸雙腺苷中多磷酸鏈的長度可能影響啤酒花腺苷酸異戊二烯基轉移酵素對其的結合與催化的活性。因此,我們進一步以點突變的方式觀察從結構模擬的結果所得可能牽涉到不同長度多磷酸雙腺苷結合的胺基酸殘基的改變是否造成酵素對不同多磷酸雙腺苷的活性發生改變。結果顯示R271A這個突變造成酵素對三磷酸雙腺苷和五磷酸雙腺苷的活性皆上升,而這也許意味著Arg271改變成Ala後減少了酵素和多磷酸雙腺苷結合的立體障礙因而使酵素對其的活性上升。綜合植物腺苷酸異戊二烯基轉移酵素(HlAIPT、AtIPT1、AtIPT3、AtIPT5、和AtIPT8)和植物四磷酸雙腺苷水解酵素(AtNUDT28/AtNUDX26和AtNUDT29/AtNUDX27)皆存在於葉綠體之中以及我們的實驗結果來看,我們認為,四磷酸雙腺苷非常可能存在於葉綠體並且很有可能作能腺苷酸異戊二烯基轉移酵素的自然受質。我們的研究不僅是探討多磷酸雙腺苷在植物中角色的開端,同時也為另一個可能具有調控功能的不尋常核苷酸衍生物開啟了一條有發展性的道路。 Diadenosine polyphosphates (ApnAs) are important signaling molecules. They are regarded as intracellular signaling molecules and involved in stress response, DNA replication and repair, and apoptosis. Moreover, out of cells, they play roles in cardiovascular system, where proliferation of vascular smooth muscle and vascular tone are affected by them, and in neuroendocrine system as one neurotransmitter. Although the occurrence of ApnAs has been demonstrated in various bacteria, fungi and animals, identification of these compounds in plants has not been reported so far. However, there are enzymes to synthesize and degrade in plant for those molecules. Those enzymes provide indirect evidence for the existence of ApnA in plant. Levels of ApnAs seem to be precisely controlled by a set of different enzymes, although their biological targets of regulation are still unclear. Data from isothermal titration calorimetry (ITC) showed that, ApnAs displayed different binding affinities to HlAIPT, and one of them is surprisingly with higher binding affinities than that of the original substrate (ATP) with an order of Ap5A > ATP > Ap4A > Ap6A > Ap3A. Compared with that of ATP, specific activities of HlAIPT to ApnA were further measured, and the order becomes ATP > Ap6A > Ap4A > Ap5A > Ap3A. These results implicated that the length of polyphosphate chain in ApnA would affect the binding and activity of HlAIPT to them. Therefore, mutations to investigate the effect of residues involving in binding of ApnA with different length based on modeling result on the activity of HlAIPT to ApnA were made. Results showed that the specific activity of R271A mutant to Ap3A and Ap5A were both increased, implying that R271A might decrease the steric hindrance and could therefore cause the increase of enzyme activity to ApnAs. Results from our study and the fact that plant AIPTs (HlAIPT, AtIPT1, AtIPT3, AtIPT5, and AtIPT8) and plant Ap4A hydrolases (AtNUDT28/AtNUDX26 and AtNUDT29/AtNUDX27) are all located in the chloroplast suggested that the existence of Ap4A in chloroplast is very possible and accordingly could be a natural substrate for AIPT. Our study here is a starting point to ask the role of ApnA in plants and might open avenues for another promising perspective of regulatory function of unusual nucleotide derivatives. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47875 |
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