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

Biochem J. 1996 May 1;315 ( Pt 3)(Pt 3):1001-5. doi: 10.1042/bj3151001.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Cattle
  • Disulfides / metabolism
  • Hydrogen-Ion Concentration
  • In Vitro Techniques
  • Isomerases / metabolism*
  • Kinetics
  • Molecular Sequence Data
  • Oligopeptides / chemistry
  • Oxidation-Reduction
  • Protein Disulfide-Isomerases
  • Spectrometry, Fluorescence
  • Substrate Specificity
  • Tryptophan / chemistry
  • Vibrio cholerae / enzymology

Substances

  • Disulfides
  • Oligopeptides
  • Tryptophan
  • Isomerases
  • Protein Disulfide-Isomerases