Mora-García S, Rodríguez-Suárez R, Wolosiuk R A
Instituto de Investigaciones Bioquímicas, Fundación Campomar, Facultad de Ciencias Exactas y Naturales-Universidad de Buenos Aires, Argentina.
J Biol Chem. 1998 Jun 26;273(26):16273-80. doi: 10.1074/jbc.273.26.16273.
Chloroplast thioredoxin-f functions efficiently in the light-dependent activation of chloroplast fructose-1, 6-bisphosphatase by reducing a specific disulfide bond located at the negatively charged domain of the enzyme. Around the nucleophile cysteine of the active site (-W-C-G-P-C-), chloroplast thioredoxin-f shows lower density of negative charges than the inefficient modulator Escherichia coli thioredoxin. To examine the contribution of long range electrostatic interactions to the thiol/disulfide exchange between protein-disulfide oxidoreductases and target proteins, we constructed three variants of E. coli thioredoxin in which an acidic (Glu-30) and a neutral residue (Leu-94) were replaced by lysines. After purification to homogeneity, the reduction of the unique disulfide bond by NADPH via NADP-thioredoxin reductase proceeded at similar rates for all variants. However, the conversion of cysteine residues back to cystine depended on the target protein. Insulin and difluoresceinthiocarbamyl-insulin oxidized the sulfhydryl groups of E30K and E30K/L94K mutants more effectively than those of wild type and L94K counterparts. Moreover, the affinity of E30K, L94K, and E30K/L94K E. coli thioredoxin for chloroplast fructose-1,6-bisphosphatase (A0.5 = 9, 7, and 3 microM, respectively) increased with the number of positive charges, and was higher than wild type thioredoxin (A0.5 = 33 microM), though still lower than that of thioredoxin-f (A0.5 = 0.9 microM). We also demonstrated that shielding of electrostatic interactions with high salt concentrations not only brings the A0.5 for all bacterial variants to a limiting value of approximately 9 microM but also increases the A0.5 of chloroplast thioredoxin-f. While negatively charged chloroplast fructose-1,6-bisphosphatase (pI = 4.9) readily interacted with mutant thioredoxins, the reduction rate of rapeseed napin (pI = 11.2) diminished with the number of novel lysine residues. These findings suggest that the electrostatic interactions between thioredoxin and (some of) its target proteins controls the formation of the binary noncovalent complex needed for the subsequent thiol/disulfide exchange.
叶绿体硫氧还蛋白-f通过还原位于叶绿体果糖-1,6-二磷酸酶带负电荷结构域的特定二硫键，在该酶的光依赖激活过程中发挥高效作用。在活性位点的亲核半胱氨酸（-W-C-G-P-C-）周围，叶绿体硫氧还蛋白-f的负电荷密度低于低效调节因子大肠杆菌硫氧还蛋白。为了研究长程静电相互作用对蛋白质二硫键氧化还原酶与靶蛋白之间硫醇/二硫键交换的贡献，我们构建了大肠杆菌硫氧还蛋白的三个变体，其中一个酸性残基（Glu-30）和一个中性残基（Leu-94）被赖氨酸取代。纯化至均一性后，通过NADP-硫氧还蛋白还原酶，NADPH对所有变体中唯一二硫键的还原速率相似。然而，半胱氨酸残基还原为胱氨酸的过程取决于靶蛋白。胰岛素和二荧光素硫代氨基甲酰胰岛素氧化E30K和E30K/L94K突变体的巯基比野生型和L94K对应物更有效。此外，E30K、L94K和E30K/L94K大肠杆菌硫氧还蛋白对叶绿体果糖-1,6-二磷酸酶的亲和力（A0.5分别为9、7和3 microM）随正电荷数量增加，且高于野生型硫氧还蛋白（A0.5 = 33 microM），尽管仍低于硫氧还蛋白-f（A0.5 = 0.9 microM）。我们还证明，用高盐浓度屏蔽静电相互作用不仅使所有细菌变体的A0.5达到约9 microM的极限值，还增加了叶绿体硫氧还蛋白-f的A0.5。带负电荷的叶绿体果糖-1,6-二磷酸酶（pI = 4.9）很容易与突变型硫氧还蛋白相互作用，而油菜籽napin（pI = 11.2）的还原速率随着新赖氨酸残基数量的增加而降低。这些发现表明，硫氧还蛋白与其（部分）靶蛋白之间的静电相互作用控制着后续硫醇/二硫键交换所需的二元非共价复合物的形成。
