DISSECTING THE MECHANISM OF PROTEIN DISULFIDE-ISOMERASE - CATALYSIS OF DISULFIDE BOND FORMATION IN A MODEL PEPTIDE

As a model for understanding how protein disulfide isomerase (PDI) cat alyzes disulfide bond formation in proteins, its action on a 28-residu e disordered peptide containing only two cysteine residues has been ex amined. Disulfide formation in the peptide using the chemical reaction with small molecule thiol/disulfide reagents, such as oxidized and re duced glutathione or cystamine and cysteamine, occurs in two steps, vi a two alternative intermediate mixed disulfides between the reagent an d either peptide cysteine residue. All thiol/disulfide forms of the pe ptide could be trapped and quantified, so the rates of their interconv ersion could be measured. Catalytic amounts of PDI increased the rates of these reactions. All rate enhancements were independent of the con centration of the peptide, indicating that it bound to PDI with an app arent K-m of less than 3 mu M. In the presence of glutathione, PDI acc elerated the formation of both single mixed disulfide species, plus th eir subsequent rearrangement to form the peptide disulfide bond, but n ot interchange of the mixed disulfide glutathione between the two cyst eine residues. In contrast, PDI did not catalyze the reaction of the r eagent cystamine with the reduced peptide to form the mixed disulfide, nor the interchange of this mixed disulfide between cysteine residues , but it did catalyze the subsequent intramolecular step of peptide di sulfide bond formation to a similar extent as with the glutathione mix ed disulfide. These effects on the two steps involving the mixed disul fides with glutathione or cystamine accounted for much of the overall catalytic effect of PDI on disulfide bond formation in the peptide, in dicating that direct transfer of disulfide bonds from PDI to the pepti de occurred less frequently. These findings demonstrate the utility of using such peptides as PDI substrates and have implications for the m echanism of action of PDI.