EFFECTS OF DSBA ON THE DISULFIDE FOLDING OF BOVINE PANCREATIC TRYPSIN-INHIBITOR AND ALPHA-LACTALBUMIN

DsbA is a protein found in the periplasm of Escherichia coli that is r equired for the formation of disulfide bonds in secreted proteins. It contains only two cysteine residues, which can form reversibly a very unstable disulfide bond that has been proposed to be the oxidant that introduces disulfide bonds into secreted proteins. The present study i nvestigates the effect of DsbA on the well-characterized disulfide-cou pled refolding processes of BPTI and of alpha-lactalbumin. Disulfide-b onded DsbA in stoichiometric amounts proved to be a very potent donor of disulfide bonds to reduced BPTI but showed little catalytic activit y at neutral pH in the presence of a glutathione redox buffer. In cont rast to the related eukaryotic enzyme protein disulfide isomerase, Dsb A did not substantially catalyze the usual intramolecular disulfide bo nd rearrangements of quasi-native folding intermediates of BPTI. Neith er did DsbA catalyze the intramolecular rearrangements observed in the three disulfide-bonded ''molten globule'' form of alpha-lactalbumin a t neutral pH. Thiol-disulfide exchange is normally very slow at acidic pH but occurs rapidly with DsbA; consequently, DsbA catalyzed the dis ulfide folding of BPTI under acidic conditions. It was then possible t o detect some increase in the rates of disulfide rearrangements, but o nly with stoichiometric amounts of DsbA and on the hour time scale. Th ese results suggest that the primary role of DsbA in the bacterial per iplasm is to introduce disulfide bonds into newly secreted proteins.