Clement-Metral J D, Höög J O, Holmgren A
Eur J Biochem. 1986 Nov 17;161(1):119-26. doi: 10.1111/j.1432-1033.1986.tb10131.x.
This paper reports the purification and characterization of a thioredoxin system (thioredoxin, thioredoxin reductase, NADPH) from the facultative phototroph Rhodobacter sphaeroides Y. Rhodobacter sph. Y thioredoxin was purified to homogeneity with an assay based on the reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by NADPH and Escherichia coli thioredoxin reductase. Rhodobacter sph. Y thioredoxin reductase was purified with the same assay using NADPH and E. coli thioredoxin. Rhodobacter sph. Y thioredoxin contained 102 amino acid residues and had a single intrachain disulfide bond. The two half-cystine residues are part of the active site made up of the sequence -Ala-Glu-Trp-Cys-Gly-Pro-Cys-Arg- which is identical to that of E. coli thioredoxin except for the presence of an Arg instead of a Lys. Rhodobacter sph. Y thioredoxin contains two tryptophan residues. The fluorescence intensity of the tryptophan residues is quenched in oxidized thioredoxin; on reduction, a much smaller increase is observed with Rhodobacter sph. Y thioredoxin than with the E. coli protein. However, the presence of 5 M guanidine X HCl results in the complete exposure of the two tryptophan residues. Rhodobacter sph. Y thioredoxin reductase has structural and functional similarities to E. coli thioredoxin reductase: it has a molecular mass of 68 kDa, and consists of two, probably identical, subunits. Each subunit has one bound FAD molecule. The enzyme is highly specific for NADPH; it is also highly specific for Rhodobacter sph. Y thioredoxin with a Km value of 3.3 +/- 0.6 microM. A kinetic study of the two thioredoxin systems shows that they have a high degree of cross-reactivity.
本文报道了兼性光合细菌球形红杆菌Y硫氧还蛋白系统（硫氧还蛋白、硫氧还蛋白还原酶、NADPH）的纯化及特性。球形红杆菌Y硫氧还蛋白通过基于NADPH和大肠杆菌硫氧还蛋白还原酶对5,5'-二硫代双（2-硝基苯甲酸）的还原作用的测定法纯化至同质。球形红杆菌Y硫氧还蛋白还原酶使用NADPH和大肠杆菌硫氧还蛋白通过相同测定法纯化。球形红杆菌Y硫氧还蛋白含有102个氨基酸残基且具有一个链内二硫键。两个半胱氨酸残基是由-Ala-Glu-Trp-Cys-Gly-Pro-Cys-Arg-序列组成的活性位点的一部分，该序列与大肠杆菌硫氧还蛋白的序列相同，只是存在一个精氨酸而非赖氨酸。球形红杆菌Y硫氧还蛋白含有两个色氨酸残基。在氧化型硫氧还蛋白中色氨酸残基的荧光强度被淬灭；还原时，与大肠杆菌蛋白相比，球形红杆菌Y硫氧还蛋白观察到的荧光强度增加要小得多。然而，5M盐酸胍的存在导致两个色氨酸残基完全暴露。球形红杆菌Y硫氧还蛋白还原酶在结构和功能上与大肠杆菌硫氧还蛋白还原酶相似：它的分子量为68kDa，由两个可能相同的亚基组成。每个亚基有一个结合的FAD分子。该酶对NADPH具有高度特异性；它对球形红杆菌Y硫氧还蛋白也具有高度特异性，Km值为3.3±0.6μM。对这两个硫氧还蛋白系统的动力学研究表明它们具有高度的交叉反应性。
