CONTRIBUTIONS OF SUBSTRATE-BINDING TO THE CATALYTIC ACTIVITY OF DSBC

DsbA and DsbC are involved in protein disulfide bond formation in the periplasm of Gramnegative bacteria. The two proteins are thought to fu lfill different functions in vivo, DsbA as a catalyst of disulfide bon d formation and DsbC as a catalyst of disulfide bond rearrangement. To explore the basis of this catalytic complementarity, the reaction mec hanism of DsbC has been examined using unstructured model peptides tha t contain only one or two cysteine residues as substrates. The reactio ns between the various forms of the peptide and DsbC occur at rates up to 10(6)-fold faster than those that involve glutathione and DsbC, an d they were constrained to occur at only one sulfur atom of disulfide bonds involving the peptide. Mixed disulfide complexes of DsbC and the peptide were 10(4)-fold more stable than the corresponding mixed disu lfides with glutathione. These observations suggest that noncovalent b inding interactions occur between the peptide and DsbC, which contribu te to the very rapid kinetics of substrate utilization. The interactio ns between DsbC and the peptide appear to be more substantial than tho se between DsbA and the same peptide. The differences in the reaction of the peptide at the active sites of DsbA and DsbC provide insight in to why DsbC is the better catalyst of disulfide bond rearrangement and how the active site chemistry of these structurally related proteins has been adapted to fulfill complementary functions.