利用單分子螢光共振能量轉移技術量測雙元體質子傳送-焦磷酸水解酶酵素活性區之距離變化

Distance Variations between Active Sites of H+-pyrophosphatase Determined by Single Molecule Fluorescence Resonance Energy Transfer

黃蘊慈(Yun-Tzu Huang)

指導教授 ： 潘榮隆

國立清華大學/生命科學院/生物資訊與結構生物研究所/博士(2010年)

摘要

質子傳送-焦磷酸水解酶(簡稱H+-PPase; EC 3.6.1.1)是維持生物體內pH恆定的重要酵素。這個獨特的質子傳送酶藉由水解生物次級代謝產物－焦磷酸產生能量以驅動質子傳送，來維持細胞膜內外的酸鹼平衡。已知此酵素的焦磷酸水解功能單位為單元體，而質子傳送功能單位為同雙元體，其每個單體的酵素活化中心均由位在第五環圈的焦磷酸結合區段與第一酸性區段，以及位在第十五環圈的第二酸性區段和數個基要胺基酸所構成。本實驗即利用單分子螢光共振能量轉移技術，量測這些重要區段及胺基酸之間的分子間距離，並觀察此酵素與受質類似物及離子結合時所發生的之距離變化。其中位在同雙元體上兩個羧酸端的距離為49.3 ± 4.0 Å，而兩個胺基端之間的距離為67.2 ± 5.7 Å。雖然兩個焦磷酸結合區段的距離相對遙遠(70.8 ± 4.8 Å)，但是當質子傳送-焦磷酸水解酶與鉀離子及焦磷酸類似物結合後，兩個焦磷酸結合區段會變得更靠近彼此(56.6 ± 4.1 Å)。此外當酵素與受質類似物結合時亦會引起同雙元體上兩個第一酸性區段及兩個H622胺基酸的距離發生重要的改變，然而此現象在鉀離子與質子傳送-焦磷酸水解酶結合時並未發生。因此，本研究在同雙元體質子傳送-焦磷酸水解酶的基要區段、重要胺基酸以及酵素活性區段之間的距離量測上提供了重要的結構意義，並且提出一個酵素與受質結合機制的模式。

關鍵字

質子傳送-焦磷酸水解酶 ； 單分子螢光共振能量轉移技術 ； 全內反射式螢光顯尾勁 ； 金奈米粒子 ； 原子力顯微鏡

並列摘要

Homodimeric H+-pyrophosphatase (H+-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel enzyme supplies energy at expense of hydrolyzing metabolic byproduct, pyrophosphate (PPi), for H+ translocation across membrane. The functional unit of a monomer suffices for enzymatic reaction of H+-PPase, while that for the translocation is homodimer. Its active site on each subunit consists of PPi binding motif, Acidic I and II motifs, and several essential residues. In this investigation, structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H+-PPase are 49.3 ± 4.0 Å and 67.2 ± 5.7 Å, respectively. Furthermore, putative PPi binding motifs on individual subunits are found to be relatively far away from each other (70.8 ± 4.8 Å), while binding of potassium and substrate analogue led them to closer proximity (56.6 ± 4.1 Å). Moreover, substrate analogue but not potassium elicits significantly distance variations between two Acidic I motifs and two H622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues and putative active sites on homodimeric subunits of H+-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H+-PPase upon substrate binding.

並列關鍵字

H+-translocating inorganic pyrophosphatase ； Single molecule fluorescence resonance energy transfer ； Total internal reflection fluorescence microscope ； Gold nanoparticle ； Atomic force microscope

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