EFFICIENT CATALYSIS OF DISULFIDE FORMATION DURING PROTEIN-FOLDING WITH A SINGLE ACTIVE-SITE CYSTEINE

Protein disulfide isomerases (PDIs) catalyze disulfide bond formation during protein folding in vivo and are essential for viability in euka ryotic cells. They share the active-site sequence C-X-X-C that forms a catalytic disulfide. The recent finding that the EUG1 protein, a PDI- related yeast protein, with C-X-X-S sequence at its active sites can c omplement PDI-deficiency raised the general question of whether disulf ide-isomerase activity is essential for cell viability or whether PDI variants with single active-site thiol groups can be catalytically act ive as disulfide isomerases. We investigated the function of the catal ytic cysteine residues in DsbA, a PDI-related protein required for dis ulfide formation in the periplasmic space of Escherichia coli, by repl acing C30 and C33 with alanine. While the mutant C30A and the double m utant CC30/33AA are inactive, C33A catalyzes disulfide-interchange rea ctions and oxidative renaturation of the reduced, unfolded thrombin in hibitor hirudin with close to wild-type efficiency Thus, the single ac tive-site thiol group of C30 is sufficient for disulfide-isomerase act ivity of the DsbA protein.