利用氫氘交換反應解構植物液泡質子傳送焦磷酸水解酶動態結構

Dynamics of the H+-translocating Pyrophosphatase Revealed by Hydrogen-Deuterium Exchange

葉庭愷(Ting-Kai Yeh)

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/碩士(2014年)

摘要

質子傳送焦磷酸水解酶（簡稱H+-PPase; EC 3.6.1.1)存在於植物細胞，細菌、古生菌、以及一些原生生物中。藉由水解生物次級代謝產物（焦磷酸)提供能量驅動質子傳送至胞腔內。近年來已經解出了此酵素的3D構型，但是對於它的動態活動仍然所知甚少。因此，我們運用了氫/氘交換反應結合質譜偵測技術觀察出H+-PPase與受質類似物結合後的動態結構改變。當蛋白質置於重水溶液中，氘原子會與蛋白主鏈上NH的氫（Backbone amide hydrogens)做交換，進而被質譜儀所偵測。在此，辨別各部位氘原子的攝取量來洞察出整體結構動態的改變。在氫氘交換結果中，與受質類似物結合後形成了較為穩固的結構，質子傳送通道緊縮，許多區段都被保護住不易被氘置換，尤其是在高保守性的酵素活性中心。另外，靠近細胞質的第一，第五及第十一環圈蓋住了上方的受質結合位，變得使基質不易進入；胞腔內側的N端與第十二環圈可能防止質子回流到細胞質中。此研究能幫助我們更加了解酵素的動態機制，有助於將來在農業生物上的應用。

關鍵字

質子傳送焦磷酸水解酶 ； 液相層析儀質譜儀 ； 電噴灑離子化 ； 氫氘交換

並列摘要

H+-translocating pyrophosphatases (H+-PPase; EC 3.6.1.1) exists in various endomembranes of plants, bacteria, and some prokaryotes. It transports H+ into lumens at the cost of hydrolyzing the ''product of anabolic reactions'', inorganic pyrophosphate (PPi). Although the crystal structure of mung bean H+-PPase has been solved recently, the motions of H+-PPase is still unclear. Here, we applied hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) to monitor the dynamic of H+-PPase between the resting (apo form) and initiated states (bound with substrate analogue). When proteins were placed in a D2O solution, the backbone hydrogens, which exchange with deuterium, would be identified by MS. Accordingly, we discerned through the deuterium uptake to insight into the structural dynamic and conformational changes. HDX on the VrH+-PPase shows a significant protection againat exchange upon binding with substrate analogue, especially in the highly conserved PPi binging motif and reveals a rigid structure to form a narrow transport pathway. Additionally, Loop1, Loop5and Loop11, in the cytosolic site, exhibit a solvent inaccessible region and form a lid to cover the substrate binding pocket; N terminal and vacuolar lumen Loop12 may prevent proton back flow into the cytosol. These results highlight the more detail understanding the mechanism of VrH+-PPase and presumably for biological and agricultural interests.

並列關鍵字

H+-translocating pyrophosphatase ； Liquid chromatography-mass spectrometry ； Electrospray ionization ； Hydrogen/deuterium exchange

參考文獻

Asaoka M, Segami S, Maeshima M. (2014). Identification of the critical residues for the function of vacuolar H+-pyrophosphatase by mutational analysis based on the 3D structure. J Biochem 46, 1-12.

Bao AK, Wang SM, Wu GQ, Xi JJ, Zhang JL, Wang CM. (2009). Overexpression of the Arabidopsis H+-PPase enhanced resistance to salt and drought stress in transgenic alfalfa (Medicago sativa L.). Plant Science 176, 232-240.

Baykov AA, Bakuleva NP, Rea PA. (1993). Steady-state kinetics of substrate hydrolysis by vacuolar H(+)-pyrophosphatase. A simple three-state model. Eur J Biochem 217, 755-762.

Belogurov GA, Lahti R. (2002). A lysine substitute for K+. A460K mutation eliminates K+ dependence in H+-pyrophosphatase of Carboxydothermus hydrogenoformans. J Biol Chem 277, 49651-4.

Bradford MM. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem 72, 248-254.