REACTIVITY AND IONIZATION OF THE ACTIVE-SITE CYSTEINE RESIDUES OF DSBA, A PROTEIN REQUIRED FOR DISULFIDE BOND FORMATION IN-VIVO

DsbA is a periplasmic protein of Escherichia coil that appears to be t he immediate donor of disulfide bonds to proteins that are secreted. I ts active site contains one accessible and one buried cysteine residue , Cys30 and Cys33, respectively, which can form a very unstable disulf ide bond between them that is 10(3)-fold more reactive toward thiol gr oups than normal. The two cysteine residues have normal properties whe n in a short peptide. In DsbA, the Cys30 thiol group is shown to be re active toward alkylating reagents down to pH 4 and to be fully ionized , on the basis of the UV absorbance of the thiolate anion at 240 nm. I ts reactivity is altered by another, unknown group on the reduced prot ein titrating with a pK(a) of about 6.7. The other cysteine residue is buried and unreactive and has a high pK(a) value. The ionization prop erties of the DsbA thiol groups can explain, at least partly, the high reactivity of its disulfide bonds and thiol groups at both neutral an d acidic pH values.