植物液泡質子輸送焦磷酸水解脢係由二個分子量為81 kDa的同元次體組成。經拓僕分析，綠豆液泡質子輸送焦磷酸水解脢包含了14個穿膜區；不同物種間液泡質子輸送焦磷酸水解脢之胺基酸序列比對發現：緊鄰於受質結合區之前（胺基端）的第五穿膜區具有很高的序列相似度。同時，親疏水分析顯示第五穿膜區之疏水性較其他穿膜區之疏水性低。因此推論，第五穿膜區可能參與了植物液泡質子輸送焦磷酸水解脢之質子輸送過程。在本論文中，我們採用定點突變法測試第五穿膜區內各個胺基酸的功能：穿膜區內的各個胺基酸分別被單點突變成丙氨酸，接著這些突變的cDNA被大量表現於酵母菌之液泡膜上，然後我們將酵母菌液泡膜單離出來，分別測定各個突變後之液泡質子輸送焦磷酸水解脢，檢視其焦磷酸水解活性及質子輸送活性的變化。結果發現，第五穿膜區內的突變可區分為二類：第一類突變之酵素活性改變較小，而第二類突變包含E225A、GYG模序（229至231）、A238S及R242A，其焦磷酸水解活性、質子輸送活性及耦合效率都出現明顯的下降。其中Y230A突變更進一步顯示對原有之陽離子效應的緩解。根據進一步的胺基酸序列比對結果，我們推論GYG模序可能是液泡質子輸送焦磷酸水解脢中的陽離子結合片段；第五穿膜區的新模型：GYG模序為中心，連接了穿膜區5A及5B，於焉成形。我們製備了Y230S及Y230F兩個突變以進一步研究酪氨酸230之結構╱功能角色；將酪氨酸突變成苯丙氨酸並不改變此酵素之焦磷酸水解活性，然而其質子輸送活性及耦合效率卻下降了百分之四十，顯見酪氨酸230上的氫氧基直接參與了液泡質子輸送焦磷酸水解脢之質子輸送過程。

Abstract
Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase containing a single type of polypeptide with a molecular mass of approximately 81 kDa. Topological analysis tentatively predicts that mung bean V-PPase contains 14 transmembrane domains. Alignment analysis of V-PPase demonstrates that the transmembrane domain 5 (TM5) of the V-PPase is highly conserved and located at the N-terminal side of the putative substrate-binding loop. Furthermore, the hydropathic analysis of V-PPase showed a relatively lower degree of hydrophobicity in TM5 region than others. Accordingly, it is believed that the TM5 is probably involved in the proton translocation of V-PPase. In this study, we used the site-directed mutagenesis to examine the functional role of amino acid residues in TM5 of V-PPase. A series of mutants singly replaced by alanine residues along TM5 was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Our results indicate that the several mutants, attributed as Group 1, displayed minor variations in enzymatic properties, while the others, attributed as Group 2 including those mutated at E225, a GYG motif (residues from 229 to 231), A238, and R242, showed a serious decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase. The mutant Y230A also displayed a relief in the cation effects on the V-PPase. Mutation of several residues along TM5 brought as well about the structural changes of V-PPase. Analysis on sequence alignment suggests that GYG motif may form a cation-binding cage for V-PPase. A model with two helical wheels on both sides of GYG motif is thus proposed for TM5 of V-PPase. Two more mutations of Y230 (Y230S and Y230F) were constructed to investigate the structure/function role of this tyrosine residue in detail. The substitution of tyrosine to phenylalanine at 230 did not change the hydrolytic activity of V-PPase much, but shifted the proton translocating ability, hence the coupling efficiency of V-PPase to 60% of the wild type. The hydroxyl moiety on the Y230 residue might be therefore directly involved in the proton translocating process of V-PPase.

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