Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli.
The redox properties of periplasmic protein disulfide isomerase (DsbA) from Escherichia coli were analyzed by measuring the equilibrium constant of the oxidation of reduced DsbA by oxidized glutathione. The experiments are based on the finding that the intrinsic tryptophan fluorescence of DsbA increases about threefold upon reduction of the enzyme, which can be explained by the catalytic disulfide bridge quenching the fluorescence of a neighboring tryptophan residue. From the specific fluorescence of DsbA equilibrated in the presence of different ratios of reduced and oxidized glutathione at pH 7, an equilibrium constant of 1.2 x 10(-4) M was determined, corresponding to a standard redox potential (E'0) of DsbA of -0.089 V. Thus, DsbA is a significantly stronger oxidant than cytoplasmic thioredoxins and its redox properties are similar to those of eukaryotic protein disulfide isomerase. The equilibrium constants for the DsbA/glutathione equilibrium were found to be strongly dependent on pH and varied from 2.5 x 10(-3) M to 3.9 x 10(-5) M between pH 4 and 8.5. The redox state-dependent fluorescence properties of DsbA should allow detailed physicochemical studies of the enzyme as well as the quantitative determination of the oxidized protein by fluorescence titration with dithiothreitol and open the possibility to observe bacterial protein disulfide isomerase "at work" during catalysis of oxidative protein folding.
Wunderlich
M
M
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Glockshuber
R
R
D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005453:Fluorescence; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D007535:Isomerases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
D004220:Disulfides; D011994:Recombinant Proteins; D014364:Tryptophan; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D004229:Dithiothreitol; D019803:Glutathione Disulfide