A Positive Charge at Position 33 of Thioredoxin Primarily Affects Its Interaction with Other Proteins but Not Redox Potential

Tiao-Yin Lin*, Ton Seng Chen

*此作品的通信作者

研究成果: Article同行評審

14 引文 斯高帕斯(Scopus)

摘要

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 Km, with little effect on kcat. 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 fl was tested. The efficiency of plating (EOP) of T3/7 on the mutant strain decreases 5 times at 37°C and 3 × 104 times at 42°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 fl by 8-fold at 37°C and 1.5-fold at 42°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.

原文English
頁(從 - 到)945-952
頁數8
期刊Biochemistry
43
發行號4
DOIs
出版狀態Published - 3 二月 2004

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