Mössner E, Huber-Wunderlich M, Rietsch A, Beckwith J, Glockshuber R, Aslund F
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland.
J Biol Chem. 1999 Sep 3;274(36):25254-9. doi: 10.1074/jbc.274.36.25254.
The thioredoxin superfamily consists of enzymes that catalyze the reduction, formation, and isomerization of disulfide bonds and exert their activity through a redox active disulfide in a Cys-Xaa(1)-Xaa(2)-Cys motif. The individual members of the family differ strongly in their intrinsic redox potentials. However, the role of the different redox potentials for the in vivo function of these enzymes is essentially unknown. To address the question of in vivo importance of redox potential for the most reducing member of the enzyme family, thioredoxin, we have employed a set of active site variants of thioredoxin with increased redox potentials (-270 to -195 mV) for functional studies in the cytoplasm of Escherichia coli. The variants proved to be efficient substrates of thioredoxin reductase, providing a basis for an in vivo characterization of NADPH-dependent reductive processes catalyzed by the thioredoxin variants. The reduction of sulfate and methionine sulfoxide, as well as the isomerization of periplasmic disulfide bonds by DsbC, which all depend on thioredoxin as catalyst in the E. coli cytoplasm, proved to correlate well with the intrinsic redox potentials of the variants in complementation assays. The same correlation could be established in vitro by using the thioredoxin-catalyzed reduction of lipoic acid by NADPH as a model reaction. We propose that the rate of direct reduction of substrates by thioredoxin, which largely depends on the redox potential of thioredoxin, is the most important parameter for the in vivo function of thioredoxin, as recycling of reduced thioredoxin through NADPH and thioredoxin reductase is not rate-limiting for its catalytic cycle.
硫氧还蛋白超家族由催化二硫键还原、形成和异构化的酶组成，这些酶通过Cys-Xaa(1)-Xaa(2)-Cys基序中的氧化还原活性二硫键发挥其活性。该家族的各个成员在其固有氧化还原电位上有很大差异。然而，这些不同氧化还原电位对这些酶体内功能的作用基本上是未知的。为了解决氧化还原电位对该酶家族中还原性最强的成员硫氧还蛋白体内重要性的问题，我们采用了一组硫氧还蛋白活性位点变体，其氧化还原电位有所增加（-270至-195 mV），用于在大肠杆菌细胞质中进行功能研究。这些变体被证明是硫氧还蛋白还原酶的有效底物，为硫氧还蛋白变体催化的NADPH依赖性还原过程的体内表征提供了基础。硫酸盐和甲硫氨酸亚砜的还原，以及周质二硫键由DsbC进行的异构化，在大肠杆菌细胞质中这些过程都依赖硫氧还蛋白作为催化剂，在互补试验中证明与变体的固有氧化还原电位密切相关。通过使用硫氧还蛋白催化NADPH还原硫辛酸作为模型反应，在体外也可以建立相同的相关性。我们提出，硫氧还蛋白直接还原底物的速率在很大程度上取决于硫氧还蛋白的氧化还原电位，这是硫氧还蛋白体内功能的最重要参数，因为通过NADPH和硫氧还蛋白还原酶使还原型硫氧还蛋白再循环对其催化循环不是限速步骤。
