Isolation and characterization of a protease-nicked thioredoxin.
An altered form of thioredoxin, composed of two peptides with molecular weights of 7 and 5 X 10(3) has been isolated from Escherichia coli B after chromatography on Ultrogel AcA54. The position of the clip site, determined by amino acid sequencing to lie between Pro(64) and Gly(65), is consistent with a cleavage within the hinge region connecting the two prominent folding domains of thioredoxin. The NH2-terminal domain containing the active site, and the carboxyl-terminal domain correspond to the 7 and 5 X 10(3) fragments, respectively. The two peptides combine to form a tight complex, T'12. At concentrations below 10(-6) M, the clipped species was not efficiently reduced by thioredoxin reductase, showing only half the activity of native thioredoxin at a protein concentration of 9 X 10(-8) M. The Km of the clipped species for thioredoxin reductase (2.9 X 10(-6) M) was nearly equivalent to that of native thioredoxin (2.2 X 10(-6) M), and the Vmax for both native and clipped thioredoxins were nearly identical. These data are taken to imply a reversible association of the fragments to produce a thioredoxin equivalent to the intact species in its interaction with thioredoxin reductase. Some evidence exists for an unfolding of the 7 X 10(3) fragment upon dissociation of the complex. A functional role for the two-domain structure of thioredoxin is proposed in which the carboxyl domain serves to stabilize the active site within the amino domain and confer the species specificity in the binding of thioredoxin to thioredoxin reductase.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification; D004228:Dithionitrobenzoic Acid; D004926:Escherichia coli / Q000032:analysis*; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000302:isolation & purification
D001426:Bacterial Proteins; D010446:Peptide Fragments; D013879:Thioredoxins; D004228:Dithionitrobenzoic Acid; D013880:Thioredoxin-Disulfide Reductase; D010447:Peptide Hydrolases