Identification of Essential Lysines Involved in Substrate Binding of Vacuolar H+-Pyrophosphatase

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研究生：	李建賢 Lee, ien-Hsien
論文名稱：	Identification of Essential Lysines Involved in Substrate Binding of Vacuolar H+-Pyrophosphatase 液泡焦磷酸水解酵素中必要離胺酸參與受質接合之鑑定
指導教授：	潘榮隆 Pan, ng-Long
口試委員:	林彩雲 Lin, Tsai-Yun 高茂傑 Kao, Mou-Chieh 蕭義勇 Hsiao, Yi-Yuong 張文綺 Chang, n-Chi 潘榮隆 Pan, ng-Long
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	60
中文關鍵詞：	H+-pyrophosphatase 、Lysine 、Chemical modification 、Site-directed mutagenesis
外文關鍵詞：	質子傳送焦磷酸水解酵素, 離胺酸, 化學修飾, 定點突變
相關次數：	點閱：2 下載：0
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H+-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) drives proton pumping against an electrochemical potential gradient by hydrolyzing pyrophosphate (PPi) to generate proton motive force for secondary transport and storage of metabolites, ions and even toxins. This unique proton pump, H+-PPase, primarily found in various endomembranes of higher plants, bacteria, and some protists, By sequence alignment there are seven highly conserved lysines in this enzyme, and, for the most part, six in cytosolic side. For realizing the functional roles of these lysines, we investigated the 18 proposed cytosolic lysine residues in mung bean H+-PPase, each of which was firstly substituted to an alanine by site-directed mutagenesis. Construction of mutants that each had a cytosolic, highly conserved lysine substituted with an alanine resulted in dramatic drops in both PPi hydrolytic and PPi-dependent H+-translocating activity. The effects caused by ions on the activities of WT and mutant H+-PPases suggest that Lys-730 may be in close proximity to the Mg2+ binding site, and the great resistance of the K694A and K695A mutants to fluoride inhibition implicates that these lysines are present in the active site. The modifier fluorescein 5’-isothiocyanate (FITC), which is used to labeling lysine residue(s) in a protein, targeted a lysine at the H+-PPase active site, but did not inhibit the hydrolytic activities of K250A, K250N, K250T, and K250S mutants in inhibition experiment, which suggested that Lys-250 is essential for substrate binding. Moreover, the decreased coupling ratio of these mutants suggested Lys-250 may be involved in proton translocation. Analysis of tryptic digests indicated that Lys-711 and Lys-717 help maintain the conformation of the active site in enzyme-substrate complex. Proteolytic evidence also demonstrated that Lys-250 is the primary target of trypsin and confirmed its crucial role in H+-PPase hydrolysis. A working model is proposed to elucidate the structural mapping of essential lysines in H+-PPase and their possible functional roles.

質子傳送焦磷酸水解酵素(簡稱H+-PPase; EC 3.6.1.1)，藉由水解焦磷酸(PPi)所產生的能量，來傳送質子，提高質膜兩側的電化學梯度差。這種獨特的質子幫浦普遍存在於高等植物的液泡膜，以及某些細菌、單細胞生物的內膜。經由蛋白質序列比對，在質子傳送焦磷酸水解酵素可以找到七個高度保留的離胺酸，而其中六個離胺酸位於蛋白質突出於細胞質的部分。為了瞭解離胺酸在質子傳送焦磷酸水解酵素中，所辦演的角色與功能，我們以綠豆液泡膜上的質子傳送焦磷酸水解酵素為研究對象，將位於細胞質部分的18個離胺酸，利用定點突變法，一一將其變為丙胺酸。高度保留的離胺酸被突變為丙胺酸後，這些突變使得質子傳送焦磷酸水解酵素的水解能力與質子傳送能力大大地減低；而透過離子效應的實驗，其數據顯示位置730的離胺酸與其輔因子，鎂離子，的接合位相當接近，此外位置694與695的離胺酸則參與在抑制因子，氟離子，的接合位上。異硫氰酸螢光素(fluorescein 5'-isothiocyanate, 簡稱FITC)，一種會與一級胺產生共價鍵結，而時常被用來標定蛋白質中的離胺酸的化學物質，已被知道能夠標定一個位於質子傳送焦磷酸水解酵素活化區的特定離胺酸。在化學修飾的實驗中，我們發現位於位置250處的突變株(K250A, K250N, K250T, K250S)，其水解焦磷酸的活性均無法被異硫氰酸螢光素所抑制，依據這一結果，我們推論位置250處的離胺酸處於受質的接合位上；且經由活性測試，這些突變株會導致耦合率下降，說明了位置250處的離胺酸亦與質子傳送效率有關。而透過胰蛋白酵素分解實驗，實驗結果顯示位置711與位置717的離胺酸，與酵素、受質形成的複合體穩定性有關，此外，此一實驗亦證明位置250處的離胺酸，正是胰蛋白酵素作用在質子傳送焦磷酸水解酵素上的重要切點，而此實驗結果亦可再次印證，位置250的離胺酸與酵素水解活性間的重要關係。我們依此建立一個活化中心結構模式，以解釋必要離胺酸在質子傳送焦磷酸水解酵素的角色。

Abbreviations ...........................................................................................8
Introduction .............................................................................................9
Experimental Procedures
Manipulation and Expression of K→A Substituted H+-PPase in Yeast Cells ........................................................................................12
Preparation of H+-PPase Enriched Yeast Microsomes ...................12
Assays of H+-PPase Activity ............................................................14
SDS-PAGE and Western Blotting Analysis .....................................15
Chemical Modification of H+-PPase by FITC ................................15
Trypsin Proteolysis ..........................................................................16
Control over Contamination of K+, Na+, and Ca2+ .........................16
Results
Heterologous Expression and Functional Characterization of the Mung Bean H+-PPase K→A Mutants .............................................17
Effects of Ions on K→A H+-PPase Mutants ....................................20
Identification of the FITC-Targeted Lysine .....................................21
Proteolysis Analysis of H+-PPase K→A Mutants ...........................22
Discussion ...............................................................................................24
References ...............................................................................................29
Table and Figures
Table 1. Effects of ions on the PPi-hydrolytic activities of WT and K→A-substituted H+-PPases ...........................................................36
Fig. 1. Topology and sequence alignment for the cytosolic lysines of mung bean H+-PPase ......................................................................38
Fig. 2. Heterologous expression of mung bean H+-PPase in yeast .................................................................................................41
Fig. 3. The expression and enzymatic activities of the mung bean H+-PPase K→A mutants .................................................................43
Fig. 4. FITC inhibition of K→A H+-PPase mutants .......................45
Fig. 5. Properties of the Lys-250 H+-PPase mutants ......................46
Fig. 6. FITC inhibition and tryptic digestion of Lys-250 H+-PPase mutants ............................................................................................48
Fig. 7. Trypsin digestion of K→A H+-PPase mutants ....................50
Fig. 8. Proposed model for essential lysines in substrate binding of H+-PPase .........................................................................................51
Appendix
Table S1. The list of primer sequences ............................................52
Fig. S1. Sequence alignment, membrane topology, and cytosolic lysines of mung bean H+-PPase ......................................................53
Fig. S2. Heterologous expression level and localization of mung bean H+-PPase mutants in yeast .....................................................58
Fig. S3. Trypsin digestion profiles of H+-PPases mutated at K250 ................................................................................................59
Fig. S4. Sequence alignment of acidic motifs of soluble- and H+-PPases .......................................................................................60

                                

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