Hirono Megumi, Nakanishi Yoichi, Maeshima Masayoshi
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Biochim Biophys Acta. 2007 Dec;1767(12):1401-11. doi: 10.1016/j.bbabio.2007.09.007. Epub 2007 Sep 29.
The H(+)-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14-17 transmembrane domains (TMs). We focused on the third quarter region of Streptomyces coelicolor A3(2) H(+)-pyrophosphatase, which contains a long conserved cytoplasmic loop. We assayed 1520 mutants for pyrophosphate hydrolysis and proton translocation, and selected 34 single-residue substitution mutants with low substrate hydrolysis and proton-pump activities. We also generated 39 site-directed mutant enzymes and assayed their activity. The mutation of 5 residues in TM10 resulted in low energy-coupling efficiencies, and mutation of conserved residues Thr(409), Val(411), and Gly(414) showed neither hydrolysis nor pumping activity. The mutation of six, five, and four residues in TM11, 12, and 13, respectively, gave a negative effect. Phe(388), Thr(389), and Val(396) in cytoplasmic loop i were essential for efficient H(+) translocation. Ala(436) and Pro(560) in the periplasmic loops were critical for coupling efficiency. These low-efficiency mutants showed dysfunction of the energy-conversion and/or proton-translocation activity. The energy efficiency was increased markedly by the mutation of two and six residues in TM9 and 12, respectively. These results suggest that TM10 is involved in enzyme function, and that TM12 regulate the energy-conversion efficiency. H(+)-pyrophosphatase might involve dynamic linkage between the hydrophilic loops and TMs through the central half region of the enzyme.
H(+)转运无机焦磷酸酶是一种水解无机焦磷酸的质子泵。它由一条含有14 - 17个跨膜结构域（TMs）的单一多肽组成。我们聚焦于天蓝色链霉菌A3(2) H(+) - 焦磷酸酶的第三个四分之一区域，该区域包含一个长的保守胞质环。我们检测了1520个突变体的焦磷酸水解和质子转运情况，筛选出34个底物水解和质子泵活性低的单残基取代突变体。我们还构建了39个定点突变酶并检测其活性。跨膜结构域10中5个残基的突变导致能量偶联效率降低，保守残基苏氨酸（409）、缬氨酸（411）和甘氨酸（414）的突变既无水解活性也无泵活性。跨膜结构域11、12和13中分别有6个、5个和4个残基的突变产生了负面影响。胞质环i中的苯丙氨酸（388）、苏氨酸（389）和缬氨酸（396）对于有效的H(+)转运至关重要。周质环中的丙氨酸（436）和脯氨酸（560）对于偶联效率至关重要。这些低效率突变体表现出能量转换和/或质子转运活性的功能障碍。跨膜结构域9和12中分别有2个和6个残基的突变使能量效率显著提高。这些结果表明跨膜结构域10参与酶的功能，跨膜结构域12调节能量转换效率。H(+) - 焦磷酸酶可能通过酶的中央半区域在亲水环和跨膜结构域之间涉及动态连接。
