Purification and properties of a recombinant sulfur analog of murine selenium-glutathione peroxidase.
We previously constructed plasmids for synthesis of glutathione-peroxidase (GPx) mutants in an Escherichia coli expression system. In these recombinant proteins either cysteine ([Cys]GPx mutant) or serine ([Ser]GPx mutant) were present in place of the active-site selenocysteine (SeCys) of the natural enzyme. We have now investigated GPx activity of [Cys]GPx and [Ser]GPx mutants. Enzyme assays performed on preparations of these partially purified proteins demonstrated that the [Cys]GPx mutant exhibited a significant GPx activity, unlike the [Ser]GPx mutant. Purification of [Cys]GPx was performed in two steps of ion-exchange chromatography giving a 98% homogenous protein in 50% yield. The purified [Cys]GPx protein was shown to be a symmetrical tetramer by the means of gel-filtration HPLC and SDS/PAGE. Two isoelectric points were found (6.8 and 7.2) which may reflect two different oxidation states of the mutant protein. The GPx activity of the [Cys]GPx mutant was optimal at pH 8.5. The [Cys]GPx mutant had a specific activity approximately 1000-fold smaller than that of the natural enzyme, and was very easily inactivated by hydroperoxides. Inhibition of the activity with iodoacetate determined a pKa of 8.3, presumably that of the active-site cysteine. Unlike that of SeGPx, the GPx activity of [Cys]GPx was only slightly inhibited by mercaptosuccinate. We discuss hypothetical mechanistic constraints of either catalytic cycle, which may explain such results.
Rocher
C
C
Centre de Recherches Roussel-UCLAF, Romainville, France.
Lalanne
J L
JL
Chaudière
J
J
D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005979:Glutathione Peroxidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D051379:Mice; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000235:genetics; D013455:Sulfur / Q000378:metabolism*
D011994:Recombinant Proteins; D012694:Serine; D013455:Sulfur; D005979:Glutathione Peroxidase; D003545:Cysteine