探討第三穿膜區於綠豆質子傳送焦磷酸水解酶扮演的功能性角色

植物質子傳送焦磷酸水解酶藉由水解焦磷酸將質子運送至液泡內，在液泡酸化上扮演重要角色。綠豆質子傳送焦磷酸水解酶包含16個穿膜區，其中第3穿膜區的親水性較其他大部分穿膜區都來得高，而且其上的胺基酸在植物中高度保守。此外對此酵素功能相當重要之內圈上的第5及6穿膜區鄰近第3穿膜區，因此第3穿膜區可能和質子傳送焦磷酸水解酶的活性相關，為了驗證此可能性，本研究利用定點突變法探討第3穿膜區在質子傳送焦磷酸水解酶上所扮演的角色。丙胺酸(絲胺酸)定點取代實驗結果顯示邊鏈朝向內圈的T138及S142可能和質子傳送效率相關。位在2個GxxxG-like功能區重要端點位置的G149/S153及G160/A164，對維持酵素活性及構形穩定是不可或缺的。此外G149鄰近區域的穩定度對蛋白質有效表達是很重要的。S153、M161、A164對於鉀離子促進質子傳送焦磷酸水解酶活性亦很重要。本研究結果顯示第3穿膜區對於質子傳送焦磷酸水解酶的焦磷酸水解、質子運送、蛋白表現及鉀離子刺激佔有必要地位。

關鍵字

質子運送焦磷酸水解酶；質子運送；穿膜區；定點突變；耦合效率； GxxxG-like 區位

並列摘要

H+-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) is critical for acidifying lumens by translocating protons across membranes at the expense of pyrophosphate (PPi) hydrolysis. Vigna radiata H+-PPase (VrH+-PPase) contains 16 transmembrane helices (TMs). The amino acids in TM3 are highly conserved in plants, and the hydrophobicity of TM3 is relatively lower than most other TMs, and. Moreover, TM5 and 6, which are the core TMs involving in H+-PPase functions are in the vicinity of TM3. It is thus speculated that TM3 is related with the H+-PPase activities. To address this possibility, site-directed mutagenesis was applied in this study to determine the role of TM3 in VrH+-PPase. Upon alanine/serine substitution, T138 and S142, whose functional groups face toward the center TMs were shown to be involved in efficient proton transport. G149/S153 and G160/A164 pairs at the critical termini of the two GxxxG-like motifs are necessary in maintaining the enzymatic activities and conformational stability. Moreover, stability in the vicinity surrounding G149 is vital for efficient expression. S153, M161, and A164 are crucial for the K+-mediated stimulation of H+-PPase. Taken together, this study demonstrates that TM3 plays essential roles in PPi hydrolysis, proton transport, expression, and K+ stimulation of H+-PPase.

並列關鍵字

Proton-translocating pyrophosphatase ； Proton transport ； Transmembrane helix ； Site-directed mutagenesis ； Coupling efﬁciency ； GxxxG-like motif

參考文獻

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探討第三穿膜區於綠豆質子傳送焦磷酸水解酶扮演的功能性角色

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