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 coli that appears to be the immediate donor of disulfide bonds to proteins that are secreted. Its active site contains one accessible and one buried cysteine residue, Cys30 and Cys33, respectively, which can form a very unstable disulfide bond between them that is 10(3)-fold more reactive toward thiol groups than normal. The two cysteine residues have normal properties when in a short peptide. In DsbA, the Cys30 thiol group is shown to be reactive 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. Its reactivity is altered by another, unknown group on the reduced protein titrating with a pKa of about 6.7. The other cysteine residue is buried and unreactive and has a high pKa value. The ionization properties of the DsbA thiol groups can explain, at least partly, the high reactivity of its disulfide bonds and thiol groups at both neutral and acidic pH values.
Nelson
J W
JW
European Molecular Biology Laboratory, Heidelberg, Germany.
Creighton
T E
TE
D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000378:metabolism; D007477:Ions; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019704:Protein Disulfide-Isomerases; D013056:Spectrophotometry, Ultraviolet
D004220:Disulfides; D007477:Ions; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D003545:Cysteine; D007460:Iodoacetamide