Ruddock, L.W. and Hirst, T.R. and Freedman, R.B. (1996) 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. Biochemical Journal, 315 . pp. 1001-1005. ISSN 0264-6021.
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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 pK(a) of the reactive cysteine residue at the active site of each enzyme. The data indicate a pK(app) of 5.6 for bovine PDI and of 5.1 for Vibrio cholerae DsbA.
|Additional information:||Part: Part 3|
|Subjects:||Q Science > QD Chemistry
Q Science > QP Physiology (Living systems) > QP506 Molecular biology
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences|
|Depositing User:||F.D. Zabet|
|Date Deposited:||07 May 2009 15:10|
|Last Modified:||11 Jun 2009 14:53|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/18771 (The current URI for this page, for reference purposes)|
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