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

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Abstract
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 re-oxidation 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.