Site-Directed Mutagenesis of Lys³⁶ in Human Thioredoxin: The Highly Conserved Residue Affects Reduction Rates and Growth Stimulation but is Not Essential for the Redox Protein's Biochemical or Biological Properties

Previous studies have demonstrated that a recombinant form of the human redox protein thioredoxin can stimulate the growth rate of Swiss 3T3 murine fibroblasts and that this ability to promote cellular proliferation was dependent upon a redox-active form. A site-directed mutagenesis study of the highly conserved Lys36 adjacent to the two active site cysteines of thioredoxin was performed to determine whether the basic residue was essential for the biochemical and mitogenic properties of human thioredoxin. Two mutants were generated in which the lysine residue was replaced with either glutamic acid (K36E) or leucine (K36L). While K36E and K36L were both redox-active in a thioredoxin-specific assay, the mutants exhibited decreased affinities for thioredoxin reductase relative to wild-type thioredoxin since their respective values increased by a factor of 5 and 7. Examination of the secondary structure of the variants by circular dichroism spectroscopy revealed that both mutants had minor variations in the overall structural content when compared to thioredoxin, with K36L being most similar to the wild-type protein. Thermal equilibrium denaturation studies of the variants showed that K36E had a T_m of 69.5 °C. A T_m value for thioredoxin and K36L could not be established because the absence of a plateau above 83 °C rendered it difficult to establish an upper base line and, hence, the T_m The two mutants were able to stimulate cellular proliferation, albeit with reduced efficiency when compared with wild-type thioredoxin. The results from this study indicate that Lys³⁶ is not essential for the biochemical or biological properties of human thioredoxin but removal of the positive charge does decrease the overall efficiency of thioredoxinmediated events.