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 KM 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 TM of 69.5 degrees C. A TM value for thioredoxin and K36L could not be established because the absence of a plateau above 83 degrees C rendered it difficult to establish an upper base line and, hence, the TM. The two mutants were able to stimulate cellular proliferation, albeit with reduced efficiency when compared with wild-type thioredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)
Oblong
J E
JE
Arizona Cancer Center, Tucson 85724.
Berggren
M
M
Gasdaska
P Y
PY
Hill
S R
SR
Powis
G
G
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D002942:Circular Dichroism; D017124:Conserved Sequence; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology
D011994:Recombinant Proteins; D013879:Thioredoxins; D004247:DNA; D008239:Lysine