Purification and properties of a recombinant sulfur analog of murine selenium‐glutathione peroxidase

Abstract

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 [CysIGPx and [SerIGPx mutants. Enzyme assays performed on preparations of these partially purified proteins demonstrated that the [CysIGPx mutant exhibited a significant GPx activity, unlike the [SerIGPx 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 p Ka, 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.