pH-dependence of the dithiol-oxidizing activity of DsbA (a periplasmic protein thiol:disulphide oxidoreductase) and protein disulphide-isomerase: studies with a novel simple peptide substrate.
A decapeptide containing two cysteine residues at positions 3 and 8 has been designed for use in monitoring the disulphide bond-forming activity of thiol:disulphide oxidoreductases. The peptide contains a tryptophan residue adjacent to one of the cysteine residues and an arginine residue adjacent to the other. Oxidation of this dithiol peptide to the disulphide state is accompanied by a significant change in tryptophan fluorescence emission intensity. This fluorescence quenching was used as the basis for monitoring the disulphide bond-forming activity of the enzymes protein disulphide-isomerase (PDI) and DsbA (a periplasmic protein thiol:disulphide oxidoreductase) in the pH range 4.0-7.5, where the rates of spontaneous or chemical oxidation are low. Reaction rates were found to be directly proportional to enzyme concentration, and more detailed analysis indicated that the rate-determining step in the overall process was the reoxidation of the reduced form of the enzyme by GSSG. The pH-dependence of the enzyme-catalysed reaction reflected primarily the pKa of the reactive cysteine residue at the active site of each enzyme. The data indicate a pKapp of 5.6 for bovine PDI and of 5.1 for Vibrio cholerae DsbA.
Ruddock
L W
LW
Research School of Biosciences, University of Kent at Canterbury, U.K.
Hirst
T R
TR
Freedman
R B
RB
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004220:Disulfides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry; D014734:Vibrio cholerae / Q000201:enzymology
D004220:Disulfides; D009842:Oligopeptides; D014364:Tryptophan; D007535:Isomerases; D019704:Protein Disulfide-Isomerases