Reactivity of Thioredoxin as a Protein Thiol-Disulfide Oxidoreductase

The reactivity of thioredoxin as a protein thiol-disulfide oxidoreductase is examined. The deprotonation of the redox active thiols in the CXXC motif is critical for the generation of the nucleophilic thiolate form that will attack the target disulfide in thiol-disulfide interchange reactions. The D26A variant was found to have only 10% of the activity of wild-type Trx-(SH) 2 toward reducing insulin disulfides. The inclusion of exogenous bases substantially increased the activities of D26N-Trx and D26L-Trx in the reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The intermolecular interactions between EcTrx and its substrate proteins leads to high affinity and substrate specificity, resulting in ∼10 4-fold enhanced rates for both the oxidation and reduction reactions at neutral pH, as compared to similar reactions with small molecule thiols and unstrained disulfides. The computational analysis indicated that the surface of EcTrx and of chloroplast Trx-m had large negatively charged areas that were absent in Trx-f.