Lin Tiao-Yin, Chen Ton-Seng
Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, Republic of China.
Biochemistry. 2004 Feb 3;43(4):945-52. doi: 10.1021/bi0355138.
Oxidoreductases of the thioredoxin superfamily possess the C-X-X-C motif. The redox potentials vary over a wide range for these proteins. A crucial determinant of the redox potential has been attributed to the variation of the X-X dipeptide. Here, we substitute Lys for Gly at the first X of Escherichia coli thioredoxin to investigate how a positive charge would affect the redox potential. The substitution does not affect the protein's redox potential. The equilibrium constant obtained from pairwise reaction between the mutant and wild-type proteins equals 1.1, indicating that the replacement does not significantly affect the thiol-disulfide redox equilibrium. However, the catalytic efficiency of thioredoxin reductase on the G33K mutant decreases approximately 2.8 times compared to that of the wild type. The mutation mainly affects K(m), with little effect on k(cat). The mutation also inhibits thioredoxin's ability to reduce insulin disulfide by approximately one-half. Whether the mutant protein supports the growth of phages T3/7 and f1 was tested. The efficiency of plating (EOP) of T3/7 on the mutant strain decreases 5 times at 37 degrees C and 3 x 10(4) times at 42 degrees C relative to that of the wild-type strain, suggesting that interaction between phage gene 5 protein and thioredoxin is hindered. The mutation also reduces the EOP of phage f1 by 8-fold at 37 degrees C and 1.5-fold at 42 degrees C. The global structure of the mutant protein does not change when studied by CD and fluorescence spectra. Therefore, G33K does not significantly affect the overall structure or redox potential of thioredoxin, but primarily interferes with its interaction with other proteins. Together with the G33D mutation, the overall results show that a charged residue at the first X has a greater influence on the molecular interaction of the protein than the redox potential.
硫氧还蛋白超家族的氧化还原酶具有C-X-X-C基序。这些蛋白质的氧化还原电位在很宽的范围内变化。氧化还原电位的一个关键决定因素归因于X-X二肽的变化。在这里，我们将大肠杆菌硫氧还蛋白第一个X位置的甘氨酸替换为赖氨酸，以研究正电荷如何影响氧化还原电位。该替换不影响蛋白质的氧化还原电位。从突变型和野生型蛋白质之间的成对反应获得的平衡常数等于1.1，表明该替换不会显著影响硫醇-二硫键的氧化还原平衡。然而，与野生型相比，硫氧还蛋白还原酶对G33K突变体的催化效率降低了约2.8倍。该突变主要影响米氏常数（K(m)），对催化常数（k(cat)）影响较小。该突变还使硫氧还蛋白还原胰岛素二硫键的能力降低了约一半。测试了突变蛋白是否支持噬菌体T3/7和f1的生长。相对于野生型菌株，T3/7在突变菌株上的平板效率（EOP）在37℃时降低5倍，在42℃时降低3×10⁴倍，这表明噬菌体基因5蛋白与硫氧还蛋白之间的相互作用受到阻碍。该突变还使噬菌体f1在37℃时的EOP降低8倍，在42℃时降低1.5倍。通过圆二色光谱（CD）和荧光光谱研究时，突变蛋白的整体结构没有变化。因此，G33K不会显著影响硫氧还蛋白的整体结构或氧化还原电位，但主要干扰其与其他蛋白质的相互作用。与G33D突变一起，总体结果表明第一个X位置的带电残基对蛋白质的分子相互作用的影响大于对氧化还原电位的影响。
