The single mutation Trp35-->Ala in the 35-40 redox site of Chlamydomonas reinhardtii thioredoxin h affects its biochemical activity and the pH dependence of C36-C39 1H-13C NMR.
The single mutation Trp35-->Ala in the 35-40 redox site of Chlamydomonas reinhardtii thioredoxin h affects its biochemical activity and the pH dependence of C36-C39 1H-13C NMR.
复制标题
莱茵衣藻硫氧还蛋白 h 35-40 氧化还原位点的单突变 Trp35-->Ala 影响其生化活性和 C36-C39 1H-13C NMR 的 pH 依赖性。
DOI：
10.1046/j.1432-1327.1998.2550185.x
复制
发表时间：
1998
期刊：
影响因子：
--
通讯作者：
J. Lancelin
中科院分区：
文献类型：
--
作者：
I. Krimm;S. Lemaire;E. Ruelland;M. Miginiac‐Maslow;Jean‐Pierre Jaquot;M. Hirasawa;D. Knaff;J. Lancelin
通过定点突变莱茵衣藻硫氧还蛋白h，研究硫氧还蛋白氧化还原位点不变的色氨酸残基的作用。尽管Trp 35-> Ala突变蛋白对NADPH-硫氧还蛋白还原酶和化学底物[二硫苏糖醇和5，5 '-二硫代-双（2-硝基苯甲酸）]仍具有氧化还原活性，但其完全丧失了激活硫醇调节的NADPH依赖性苹果酸脱氢酶的能力。然而，它能够激活突变的苹果酸脱氢酶，其中仅保留最暴露的二硫化物。在pH 5.8-10范围内，比较了野生型和突变蛋白在还原和氧化状态下的氧化还原位点Cys β 1H/13 C-NMR频率的pH依赖性。该突变不影响保守的掩埋Asp 30，其在氧化蛋白中的pKa为7.5，与以前的研究一致。然而，对于还原形式的蛋白质，两个半胱氨酸的共振的pH依赖性受到突变的强烈影响。在野生型硫氧还蛋白的情况下，发现两个表观pKa值约为7.0和9.5，并可分别归属于Cys 36和Cys 39硫醇的滴定，类似于大肠杆菌硫氧还蛋白的情况。对于突变的硫氧还蛋白，发现单个pKa约为8.3。该结果可解释为Cys 36或Cys 39或两者的单个pKa。虽然突变明显影响电离，但活性位点Cys对的测量氧化还原电位不受Trp 35->Ala突变的显著影响。芳香族侧链上的催化硫氧还蛋白中的半胱氨酸残基的反应性提出了可能的作用。
The role of the invariant Trp residue at the redox site of thioredoxins was investigated by site-directed mutagenesis of a Chlamydomonas reinhardtii thioredoxin h. Though being still redox active with NADPH-thioredoxin reductase and chemical substrates [dithiothreitol and 5,5'-dithio-bis(2-nitrobenzoic acid)] the Trp35-->Ala-mutated protein completely lost the capacity to activate the thiol-regulated NADPH-dependent malate dehydrogenase. However, it was able to activate a mutant malate dehydrogenase where only the most exposed disulfide was retained. The pH dependence of the redox-site Cys beta 1H/13C-NMR frequencies of the wild-type and mutated proteins, in both the reduced and oxidised states, were compared over the pH range 5.8-10. The mutation does not affect the conserved buried Asp30, which titrates with a pKa of 7.5 in the oxidised proteins in agreement with previous studies. However, for the reduced forms of the proteins, the pH dependence of resonances of both Cys was strongly affected by the mutation. In the case of the wild-type thioredoxin, two apparent pKa values were found around 7.0 and 9.5 and could be assigned to the titration of Cys36 and Cys39 thiol, respectively, similar to the case of Escherichia coli thioredoxin. For the mutated thioredoxin a single pKa was found around 8.3. This result can be interpreted as a single pKa of either Cys36 or Cys39 or both. While the mutation clearly affects ionisations, the measured redox potentials of the active-site Cys pair are not significantly affected by the Trp35-->Ala mutation. Possible roles of an aromatic side chain on the reactivity of the catalytic Cys residues in thioredoxins are proposed.