SITE-DIRECTED MUTAGENESIS OF LYS(36) IN HUMAN THIOREDOXIN - THE HIGHLY CONSERVED RESIDUE AFFECTS REDUCTION RATES AND GROWTH-STIMULATION BUTIS NOT ESSENTIAL FOR THE REDOX PROTEINS BIOCHEMICAL OR BIOLOGICAL PROPERTIES

Previous studies have demonstrated that a recombinant form of the huma n redox protein thioredoxin can stimulate the growth rate of Swiss 3T3 murine fibroblasts and that this ability to promote cellular prolifer ation was dependent upon a redox-active form. A site-directed mutagene sis study of the highly conserved Lys(36) adjacent to the two active s ite cysteines of thioredoxin was performed to determine whether the ba sic 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-specifi c assay, the mutants exhibited decreased affinities for thioredoxin re ductase relative to wild-type thioredoxin since their respective K-M v alues increased by a factor of 5 and 7. Examination of the secondary s tructure of the variants by circular dichroism spectroscopy revealed t hat both mutants had minor variations in the overall structural conten t when compared to thioredoxin, with K36L being most similar to the wi ld-type protein. Thermal equilibrium denaturation studies of the varia nts showed that K36E had a T-M of 69.5 degrees C. A T-M value for thio redoxin and K36L could not be established because the absence of a pla teau above 83 degrees C rendered it difficult to establish an upper ba se line and, hence, the T-M. The two mutants were able to stimulate ce llular proliferation, albeit with reduced efficiency when compared wit h wild-type thioredoxin. The results from this study indicate that Lys (36) is not essential for the biochemical or biological properties of human thioredoxin but removal of the positive charge does decrease the overall efficiency of thioredoxin-mediated events.