THIOREDOXIN-C' - MECHANISM OF NON-COVALENT COMPLEMENTATION AND REACTIONS OF THE REFOLDED COMPLEX AND THE ACTIVE-SITE CONTAINING FRAGMENT WITH THIOREDOXIN REDUCTASE

Thioredoxin-C' is a refolded complex of the two inactive fragments thioredoxin-C-(1-37) and thioredoxin-C-(38-108) obtained by CNBr cleavage at the single Met-37 of thioredoxin from Escherichia coli. Thioredoxin-C'-S2 is a weak complex that dissociates during gel chromatography, during gel electrophoresis, or in activity measurements as a substrate for NADPH and thioredoxin reductase. The association of the peptide fragments to thioredoxin-C'-S2 and its dissociation were both rapid processes with half-times of 1-2 min at 10-6 M. Variation of the relative proportion of the fragments in the assay of thioredoxin-C' with thioredoxin reductase gave an apparent kD for thioredoxin-C'-S2 of 2ËŸ10-6 M at 25 °C. Thioredoxin-C'-S2 was a good substrate for NADPH and thioredoxin reductase and was calculated to have more than 50% relative activity when compared with thio-redoxin-S2. In contrast, thioredoxin-C'-(SH)2 was inactive as a hydrogen donor for E. coli ribonucleotide reductase or as an insulin disulfide reductase, strongly suggesting that Met-37 is essential for the conformational change on oxidoreduction of thioredoxin. The active-site disulfide in thioredoxin-C-(1-37) was not reduced by NADPH and thioredoxin reductase; instead, the fragment was an inhibitor of thioredoxin reductase in the presence of NADPH and DTNB, which suggested nucleated folding of the fragment to generate a binding site for thioredoxin reductase. Structure-function relationships for chemically modified thioredoxin-C-(1-37) suggest essential functions for Met-37 and Lys-36 of thioredoxin in the interactions with thioredoxin reductase and ribonucleotide reductase. A model, based on the results with thioredoxin-C', is presented to describe the three-dimensional complementarity of thioredoxin and thioredoxin reductase. © 1979, American Chemical Society. All rights reserved.