Structure-function relationships in an anion-translocating ATPase
Structure-function relationships in an anion-translocating ATPase
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DOI：
10.1042/0300-5127:0280520
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发表时间：
2000-08-01
影响因子：
3.9
通讯作者：
Rosen, BP
中科院分区：
文献类型：
--
作者：
Bhattacharjee, H;Zhou, T;Rosen, BP
ArsAB-ATPase是一种位于大肠杆菌内膜上的外排泵。这种转运ATPase通过亚砷酸盐和亚硫酸盐从细胞中排出而对它们产生抵抗力。该泵由两个亚基组成，催化ARSA亚基和膜亚单位ARSB。该复合体在许多方面类似于ATP结合盒(‘ABC’)转运体，后者通常有两组，每组六个跨膜螺旋片段和TNO核苷酸结合域(NBD)。45 kDa的ARSB蛋白有12个跨膜片段。ARSB含有底物转运途径，能够作为阴离子转运体发挥作用。63 kDa ARSA蛋白是一种底物激活的ATPase。它有两个同源的一半，阿尔。和A2，这显然是祖先基因复制和融合的结果。每一半都有一个共识的NBD。通过分子遗传学和生化、结构和动力学分析相结合的方法阐明了ARSA-ATPase的变构激活机制，通过ARSA的单一色氨酸衍生物的停流荧光测量，可以揭示底物、激活剂和/或产物结合所产生的构象变化。结果表明，整个反应的限速步骤是酶的两种构象之间的缓慢异构化。变构激活增加了这种异构化的速度，从而使产物的释放变得限速，从而加速了催化。ABC转运蛋白表现出类似的底物激活ATPase活性，可以经历类似的构象变化来克服速率限制步骤。因此，ArsAB泵是一个有用的模型，可以用来阐明ABC超家族转运ATPase的机制。
The ArsAB ATPase is an efflux pump located in the inner membrane of Escherichia coli. This transport ATPase confers resistance to arsenite and antimonite by their extrusion from the cells. The pump is composed of two subunits, the catalytic ArsA subunit and the membrane subunit,ArsB. The complex is similar in many ways to ATP-binding cassette ('ABC') transporters, which typically have two groups of six transmembrane-spanning helical segments and tno nucleotide-binding domains (NBDs). The 45 kDa ArsB protein has 12 transmembrane-spanning segments. ArsB contains the substrate translocation pathway and is capable of functioning as an anion uniporter. The 63 kDa ArsA protein is a substrate-activated ATPase. It has two homologous halves, Al. and A2, which are clearly the result of an ancestral gene duplication and fusion. Each half has a consensus NBD. The mechanism of allosteric activation of the ArsA ATPase has been elucidated by a combination of molecular genetics and biochemical, structural and kinetic analyses, Conformational changes produced by binding of substrates, activator and/or products could be revealed by stopped-flow fluorescence measurements with single-tryptophan derivatives of ArsA. The results demonstrate that the rate-limiting step in the overall reaction is a slow isomerization between two conformations of the enzyme. Allosteric activation increases the rate of this isomerization such that product release becomes rate-limiting, thus accelerating catalysis. ABC transporters, which exhibit similar substrate activation of ATPase activity, can undergo similar conformational changes to overcome a rate-limiting step. Thus the ArsAB pump is a useful model for elucidating mechanistic aspects of the ABC superfamily of transport ATPases.