Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase
Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase
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DOI：
10.1074/jbc.m009743200
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发表时间：
2001-03-09
影响因子：
4.8
通讯作者：
Maeshima, M
中科院分区：
文献类型：
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作者：
Nakanishi, Y;Saijo, T;Maeshima, M
液泡H+转移无机焦磷酸酶(V-PPase)以PP为能量供体，需要游离的镁离子来维持酶的活性和稳定性。为了确定催化结构域，我们通过定点突变和在酵母中异源表达的方法，分析了绿豆V-PPase的PPI结合位点和两个保守的酸性区域的带电残基(ASP(253)，Lys(261)，Glu(263)，Asp(279)，Asp(283)，Asp(287)，Asp(723)，Asp(727)和Asp(731))。氨基酸残基被丙氨酸和保守残基取代后，PPI水解酶活性显著降低，H+转运活性完全丧失。与底物结合引起的V-PPase的构象变化反映在对胰酶的敏感性上，野生型V-PPase能被胰酶完全消化，但不能被mg-PPI存在，而两个V-PPase突变体K261A和E263A即使在底物存在下也变得对胰酶敏感。这些结果表明，第二个酸性区域也参与了底物的水解，至少两个残基，Lys(261)和GLU(263)是底物结合功能所必需的。根据保守突变体K261R和E263D显示部分PPI水解活性但没有质子泵活性的观察，我们推测两个残基Lys(261)和Glu(263)可能与PP从水解到H+运输的能量转换有关。从胰酶对镁离子的敏感性也表明了天冬氨酸(253)和谷氨酸(263)这两个残基在镁离子结合功能中的重要性。此外，还发现这两个酸性区域包含P型ATPase共有的基本共同基序。
Vacuolar H+-translocating inorganic pyrophosphatase(V-PPase) uses PP, as an energy donor and requires, free Mg2+ for enzyme activity and stability. To determine the catalytic domain, we analyzed charged residues (ASp(253), Lys(261), GlU(263), ASp(279), Asp(283), Asp(287), Asp(723), Asp(727), and Asp(731)) in the putative PPi-binding site and two conserved acidic regions of mung bean V-PPase by site-directed mutagenesis and heterologous expression in yeast. Amino acid substitution of the residues with alanine and conservative residues resulted in a marked decrease in PPi hydrolysis activity and a complete loss of H+ transport activity. The conformational change of V-PPase induced by the binding of the substrate was reflected in the susceptibility to trypsin, Wild-type V-PPase was completely digested by trypsin but not in the presence of Mg-PPi, while two V-PPase mutants, K261A and E263A, became sensitive to trypsin even in the presence of the substrate. These results suggest that the second acidic region is also implicated in the substrate hydrolysis and that at least two residues, Lys(261) and GLU(263), are essential for the substrate-binding function. From the observation that the conservative mutants K261R and E263D showed partial activity of PPi hydrolysis but no proton pump activity, we estimated that two residues, Lys(261) and GlU(263), might be related to the energy conversion from PP, hydrolysis to H+ transport. The importance of two residues, Asp(253) and GlU(263), in the Mg2+-binding function was also suggested from the trypsin susceptibility in the presence of Mg2+. Furthermore, it was found that the two acidic regions include essential common motifs shared among the P-type ATPases.