Arrangement of the epsilon subunit in the Escherichia coli ATP synthase from the reactivity of cysteine residues introduced at different positions in this subunit.
Arrangement of the epsilon subunit in the Escherichia coli ATP synthase from the reactivity of cysteine residues introduced at different positions in this subunit.
复制标题
根据引入该亚基不同位置的半胱氨酸残基的反应性，得出大肠杆菌 ATP 合酶中 epsilon 亚基的排列。
DOI：
10.1016/0005-2728(95)00040-p
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Capaldi,RA
中科院分区：
文献类型：
--
作者：
Aggeler,R;Weinreich,F;Capaldi,RA
ecf1f0是从三个突变体中纯化出来的，其中一个Cys通过位点定向诱变在柱亚基中加入：这些突变体分别是ϵS10C， ϵH38C和ϵS108C。来自突变体ϵS10C的ecf1f0酶的活性比野生型酶高2倍，这是由于ε亚基与复合物其余部分的关联发生了改变，但仍表现出正常的质子泵功能。另外两个突变体的atp酶活性与野生型相似。引入的Cys在ϵS10C和ϵS108C中与马来酰亚胺进行了反应。在ϵH38C中，引入的Cys在分离的ECF1中很容易与n -乙基马来酰亚胺反应，但在ECF1F0中无法与该或其他马来酰亚胺反应。当这个位于38位的Cys与分离的ecf1中的各种马来酰亚胺反应，然后ecf1结合回F0时，两部分之间的相互作用受到干扰。虽然用未修饰的ecf1重组的ecf1f0功能正常，但与马来酰亚胺反应的Cys-38酶显示出大大减少的质子泵，对DCCD的抑制作用仅为未修饰或野生型酶的50%左右，并且具有更高的LDAO激活（高达8.3倍，野生型为4倍）。先前在突变体ϵS10C和ϵS108C的ecf1研究中观察到核苷酸依赖性构象变化。当检查来自这些突变体的完整ecf1f0时，在与四氟苯肼马来酰亚胺交联实验中观察到相同的核苷酸依赖性结构变化。综上所述，Cys反应性数据和交联结果提供了酶复合物中柱亚基的方向。
ECF1F0has been purified from three mutants in which a Cys has been incorporated by site-directed mutagenesis in the ϵ subunit: these mutants are ϵS10C, ϵH38C and ϵS108C, respectively. ECF1F0from the mutant ϵS10C had a 2-fold higher activity than wild-type enzyme, due to altered association of the ϵ subunit with the rest of the complex, and yet showed normal proton pumping function. The other two mutants had ATPase activities similar to wild-type enzyme. The introduced Cys was exposed for reaction with maleimides in ϵS10C and ϵS108C. In ϵH38C, the introduced Cys reacted readily with N-ethylmaleimide in isolated ECF1, but was unavailable for reaction with this or other maleimides in ECF1F0. When this Cys at position 38 in the ϵ subunit was reacted with various maleimides in isolated ECF1and then the ECF1bound back to F0, the interaction between the two parts was perturbed. While ECF1F0reconstituted with unmodified ECF1functioned normally, enzyme with maleimide-reacted Cys-38 showed much reduced proton pumping, had only around 50% of the DCCD inhibition of unmodified or wild-type enzyme, and had a much higher LDAO activation (as much as 8.3-fold, c.f. 4-fold for wild type). Nucleotide-dependent conformational changes have been observed previously, in studies of ECF1from the mutants ϵS10C and ϵS108C. Identical nucleotide-dependent structural changes were observed in cross-linking experiments with tetrafluorophenylazide maleimides when the intact ECF1F0from these mutants was examined. Taken together, the Cys reactivity data and cross-linking results provide the orientation of the ϵ subunit in the enzyme complex.