A positive charge at position 33 of thioredoxin primarily affects its interaction with other proteins but not redox potential.
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.
Lin
Tiao-Yin
TY
Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, Republic of China. tylin@cc.nctu.edu.tw
Chen
Ton-Seng
TS
D019943:Amino Acid Substitution / Q000235:genetics; D017903:Bacteriophage T3 / Q000254:growth & development; D017123:Bacteriophage T7 / Q000254:growth & development; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology / Q000821:virology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D017912:Inovirus / Q000254:growth & development; D007328:Insulin / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D004220:Disulfides; D029968:Escherichia coli Proteins; D007328:Insulin; D013879:Thioredoxins; D009249:NADP; D008239:Lysine; D005998:Glycine