Identification of a region involved in the communication between the NADP(H) binding domain and the membrane domain in proton pumping E-coli transhydrogenase
Identification of a region involved in the communication between the NADP(H) binding domain and the membrane domain in proton pumping E-coli transhydrogenase
复制标题
DOI：
10.1021/bi0103157
复制
发表时间：
2001-08-21
期刊：
影响因子：
2.9
通讯作者：
Rydström, J
中科院分区：
文献类型：
--
作者：
Althage, M;Bizouarn, T;Rydström, J
大肠杆菌转氢酶的两个亲水区I和III分别与NAD(H)和NADP(H)结合，位于膜的胞液侧，而疏水区II由13个跨膜α螺旋组成，负责质子的转运。在目前的研究中，连接结构域II和III的片段βC260-βS266的特征主要是因为它在转氢酶反应的生物能量偶联中扮演了角色。该片段的每个残基都被无半胱氨酸背景中的半胱氨酸取代，并对突变的蛋白质进行了分析。除βS266C外，荧光马来酰亚胺衍生物MIANS与βC260-βR266区域的每个半胱氨酸的结合研究表明，当NADP(H)与结构域IH结合时，可及性增加；而βS266的作用则相反。分离到一个以氧化为主的β213-βR265双半胱氨酸突变体，这表明野生型酶中相应的残基位置很近，形成了盐桥。βS260C、βK261C、βA262C、βM263和βN264突变体对质子偶联反应有明显的抑制作用。同样，一些βR265突变体和β213C突变体表现出抑制质子偶联反应，但也显著增加了K-m(NADPH)值。结论：可移动铰链区βC260-βS266和βD213-βR265盐桥在结构域II的质子转位/结合事件与结构域III的NADP(H)结合/释放之间的联系中起着至关重要的作用。
The two hydrophilic domains I and III of Escherichia coli transhydrogenase containing the binding sites for NAD(H) and NADP(H), respectively, are located on the cytosolic side of the membrane, whereas the hydrophobic domain II is composed of 13 transmembrane alpha -helices, and is responsible for proton transport. In the present investigation the segment beta C260-beta S266 connecting domain II and III was characterized primarily because of its assumed role in the bioenergetic coupling of the transhydrogenase reaction. Each residue of this segment was replaced by a cysteine in a cysteine-free background, and the mutated proteins analyzed. Except for beta S266C, binding studies of the fluorescent maleimide derivative MIANS to each cysteine in the beta C260-beta R266 region revealed an increased accessibility in the presence of NADP(H) bound to domain IH; an opposite effect was observed for beta S266. A beta 213-beta R265 double cysteine mutant was isolated in a predominantly oxidized form, suggesting that the corresponding residues in the wild-type enzyme are closely located and form a salt bridge. The beta S260C, beta K261C, beta A262C, beta M263, and beta N264 mutants showed a pronounced inhibition of proton-coupled reactions. Likewise, several beta R265 mutants and the beta 213C mutant showed inhibited proton-coupled reactions but also markedly increased K-m(NADPH) values. It is concluded that the mobile hinge region beta C260-beta S266 and the beta D213- beta R265 salt bridge play a crucial role in the communication between the proton translocation/binding events in domain II and binding/release of NADP(H) in domain III.