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

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. (The full text of this publication is not available from this repository)

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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 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.

Item Type: Article
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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