TRANSPORT ATPASE--ENERGY COUPLING

该质粒决定了水稻对砷和锑化合物的抗性 大肠埃希氏菌是由阴离子转运酶介导的。这 ATPase不属于任何已知的溶质类别- 移位ATPase。它由一个完整的膜蛋白ARSB组成 其功能是载体，以及外周膜蛋白ARSA 形成催化亚单位。在相互作用时，ARSA和ARSB 蛋白质形成一个功能泵，将阴离子挤出细胞外。 拟议研究的长期目标是了解 ATP水解的能量与阴离子的转移通过 ARS ATPase。 催化成分ARSA是一种阴离子刺激的ATPase，它 在阴离子存在的情况下进行二聚反应。ARSA的每个单体都有 两个同源的核苷酸结合域，似乎是一个 反式结构域在两个多肽链之间的相互作用导致 界面的形成。我们将检验这一假设，即存在 在ARSA蛋白的同源二聚体中有两个活性部位，每个部位 是由两个多肽链的残基组成的。的组成 活性部位将通过杂交方法进行检测，其中 两个失活点突变蛋白的混合--每个蛋白都有一个缺陷 核苷酸结合结构域-预计会导致一种 活动站点。野生型蛋白与野生型蛋白的负互补 两个结构域都有缺陷的突变将导致ATPase活性丧失 如果活性中心是由两者之间共享的残基形成的 多肽链。我们将确定亚单位之间的接触地点 并通过体外诱变改变这些区域的残基以确定 这些相互作用在泵的整体功能中的作用。 三磷酸腺苷的类似物将用于研究环境和催化 两个ATP结合位点的性质，这将是关键的 了解能量转导的机制。可能的 核苷酸结合区的相互作用将与F1相关 质子转移ATPase，其中第一位的催化速率是 通过将核苷酸与第二个结合而增加了几倍 第三个地点。 该泵在结构和功能上与ABC成员相似 类型转运蛋白，如P-糖蛋白和CFTR蛋白 哺乳动物细胞。因此，这些研究将使我们深入了解 Ars-ATPase与多种分子间能量偶联的分子机制 其他离子和溶质转运泵。

The plasmid determined resistance to arsenic and antimony compounds in Escherichia coli is mediated by an anion-translocating ATPase. This ATPase does not fall into any of the known classes of the solute- translocating ATPases. It consists of an integral membrane protein ArsB that functions as a carrier, and a peripheral membrane protein ArsA that forms the catalytic subunit. Upon interaction, the ArsA and the ArsB proteins form a functional pump to extrude the anions out of the cell. The long term objective of the proposed research is to understand how the energy of ATP hydrolysis is coupled to the translocation of anions by the Ars ATPase. The catalytic component ArsA is an anion-stimulated ATPase and it undergoes dimerization in presence of the anion. Each monomer of ArsA has two homologous nucleotide binding domains, and it appears that an interaction of domains in trans between two polypeptide chains results in the formation of an interface. We will test the hypothesis that there are two active sites in the homodimer of the ArsA protein, and that each site is composed of residues from two polypeptide chains. The composition of the active sites will be examined by a hybridization approach, where mixing of two inactive point mutant proteins- each defective in one nucleotide binding domain- is expected to result in generation of one active site. Negative complementation between the wild type protein and a mutant defective in both domains will result in loss of ATPase activity if the active site is formed of shared residues between the two polypeptide chains. We will identify sites of contact between the subunits and alter residues in those regions by in vitro mutagenesis to determine the role of these interactions in the overall functioning of the pump. Analogs of ATP will be used to study the environment and the catalytic nature of the two ATP binding sites, which will be critical for understanding the mechanism of energy transduction. The possible interaction of nucleotide binding domains will have relevance to the F1 proton translocating ATPase, where rate of catalysis at the first site is increased several fold by binding of nucleotide to the second and the third site. This pump bears structural and functional similarity to members of the ABC type transporters such as the P-glycoprotein and the CFTR protein in the mammalian cells. Hence, these studies will give an insight into the molecular mechanism of energy coupling by Ars ATPase and by a variety of other ion and solute translocating pumps.