Van Ru C, Pan Yih J, Hsu Shen H, Huang Yun T, Hsiao Yi Y, Pan Rong L
Department of Life Sciences and Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University, Hsin Chu 30043, Taiwan, Republic of China.
Biochim Biophys Acta. 2005 Aug 15;1709(1):84-94. doi: 10.1016/j.bbabio.2005.05.011.
Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase consisting of 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 demonstrated that the transmembrane domain 5 (TM5) of the enzyme is highly conserved in plants and located at the N-terminal side of the putative substrate-binding loop. The hydropathic analysis of V-PPase showed a relatively lower degree of hydrophobicity in the TM5 region as compared to other domains. Accordingly, it appears that TM5 is probably involved in the proton translocation of V-PPase. In this study, we used 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 were constructed and over-expressed in Saccharomyces cerevisiae; they were then used to determine their enzymatic activities and proton translocations. Our results indicate that several mutants displayed minor variations in enzymatic properties, while others 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. Moreover, the mutation at Y230 relieved several cation effects on the V-PPase. The GYG motif presumably plays a significant role in maintaining structure and function of V-PPase.
液泡H⁺转运无机焦磷酸酶（V-PPase；EC 3.6.1.1）是一种同型二聚体质子转运酶，由一种分子量约为81 kDa的单一类型多肽组成。拓扑分析初步预测绿豆V-PPase含有14个跨膜结构域。V-PPase的比对分析表明，该酶的跨膜结构域5（TM5）在植物中高度保守，位于假定的底物结合环的N端。V-PPase的亲水性分析表明，与其他结构域相比，TM5区域的疏水性程度相对较低。因此，TM5似乎可能参与了V-PPase的质子转运。在本研究中，我们使用定点诱变来研究V-PPase的TM5中氨基酸残基的功能作用。构建了一系列沿TM5被丙氨酸残基单个取代的突变体，并在酿酒酵母中过表达；然后用它们来测定其酶活性和质子转运。我们的结果表明，几个突变体在酶学性质上表现出微小变化，而其他突变体，包括在E225、GYG基序（229至231位残基）、A238和R242处突变的那些突变体，显示出V-PPase的酶活性、质子转运和偶联效率严重下降。此外，Y230处的突变减轻了几种阳离子对V-PPase的影响。GYG基序可能在维持V-PPase的结构和功能中起重要作用。
