Role of Tryptophan Residues in the Clostridium tetani H+-PPase Determined by Site-directed Mutagenesis

質子傳送-焦磷酸水解酶是一種質子傳送幫浦，運用了簡單的無機小分子—焦磷酸做為能量的來源，來驅動質子通過細胞膜。從破傷風桿菌中發現的質子傳送-焦磷酸水解酶擁有十六個跨膜結構，而且在整個蛋白質中只發現了三個色氨酸。色氨酸是一種擁有芳香族環的氨基酸，同時對許多蛋白質來說是很重要的氨基酸。因此，我們藉由色氨酸定點取代突變法來研究色氨酸在破傷風桿菌中質子傳送-焦磷酸水解酶的角色。首先，我們先測定經由色氨酸定點取代突變法所突變的質子傳送-焦磷酸水解酶中的焦磷酸水解活性、傳送質子的能力、及質子偶合效率。用組氨酸取代在75 位置上的色氨酸後，發現了質子傳送能力大幅地下降，這表示說，蛋白質在結構上有明顯的改變。再者，我們測量了一些常見的離子 (K+、Ca2+、Na+、F-)在突變後質子傳送-焦磷酸水解酶中的影響。添加鈣離子、鈉離子、氟離子及抽離鉀離子，皆會造成野生株及突變株某種程度上質子水解活性的下降。破傷風桿菌中質子傳送-焦磷酸水解酶的功能及反應機制在本研究中能獲得相應的闡明。

關鍵字

定點突變法；破傷風桿菌；質子傳送-焦磷酸水解酶；色氨酸

並列摘要

H+-translocating inorganic pyrophosphatase (H+-PPase), a member of proton pumps, utilizes inorganic pyrophosphate (PPi) as energy source to generate proton-motive forces across membranes. H+-PPase from Clostridium tetani (CtH+-PPase) contains 16 transmembrane domains and shows only three tryptophan residues in entire protein. Tryptophan is an important aromatic amino acid for many proteins. Therefore, we investigated the role of tryptophan residues in CtH+-PPase by means of tryptophan-substitution mutagenesis. The PPi hydrolysis activities, proton translocations, and coupling efficiencies of the mutated CtH+-PPases were then determined. Substitution for W75 showed a dramatic decrease in coupling ratio, indicating obvious structural changes of the protein. Furthermore, the effect of common ions (K+, Ca2+, Na+, F-) added to these mutated CtH+-PPases were measured. Addition of Ca2+, Na+, F- and subtraction of K+ would diminish the PPi hydrolysis activities in wild type and mutants to a certain extent. The function and conformational change of CtH+-PPase were accordingly elucidated in this study.

並列關鍵字

Site-directed Mutagenesis ； Clostridium tetani ； H+-PPase ； Tryptophan

參考文獻

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國際替代計量

Role of Tryptophan Residues in the Clostridium tetani H+-PPase Determined by Site-directed Mutagenesis

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