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.

[1]  C. Bénicourt,et al.  Cloning of murine SeGpx cDNA and synthesis of mutated GPx proteins in Escherichia coli. , 1991, Gene.

[2]  W. Koppenol.  Oxyradical reactions: from bond‐dissociation energies to reduction potentials , 1990, FEBS letters.

[3]  R. Sunde.  Molecular biology of selenoproteins. , 1990, Annual review of nutrition.

[4]  B. Ames,et al.  An alkyl hydroperoxide reductase from Salmonella typhimurium involved in the defense of DNA against oxidative damage. Purification and properties. , 1989, The Journal of biological chemistry.

[5]  W. Leinfelder,et al.  Gene for a novel tRNA species that accepts L-serine and cotranslationally inserts selenocysteine , 1988, Nature.

[6]  A. Böck,et al.  Nucleotide sequence and expression of the selenocysteine-containing polypeptide of formate dehydrogenase (formate-hydrogen-lyase-linked) from Escherichia coli. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[7]  P. Harrison,et al.  The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the ‘termination’ codon, TGA. , 1986, The EMBO journal.

[8]  B. Ames,et al.  Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium , 1985, Cell.

[9]  A. Wendel,et al.  A novel biologically active seleno-organic compound--II. Activity of PZ 51 in relation to glutathione peroxidase. , 1984, Biochemical pharmacology.

[10]  A. Tappel,et al.  Interaction of gold(I) with the active site of selenium-glutathione peroxidase. , 1984, Journal of inorganic biochemistry.

[11]  A. Tappel,et al.  Mechanism of selenium-glutathione peroxidase and its inhibition by mercaptocarboxylic acids and other mercaptans. , 1984, The Journal of biological chemistry.

[12]  L. Flohé,et al.  The amino-acid sequence of bovine glutathione peroxidase. , 1984, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[13]  A. Tappel,et al.  Identification of a selenocysteine-specific aminoacyl transfer RNA from rat liver. , 1982, Biochimica et biophysica acta.

[14]  D. Hatfield,et al.  Opal suppressor serine tRNAs from bovine liver form phosphoseryl-tRNA. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[15]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[16]  H. Ganther,et al.  32 – Selenium and Glutathione Peroxidase in Health and Disease—A Review , 1976 .

[17]  L. Flohé,et al.  Glutathione peroxidase: A selenoenzyme , 1973, FEBS letters.

[18]  H. Ganther,et al.  Selenium: Biochemical Role as a Component of Glutathione Peroxidase , 1973, Science.

[19]  L. Flohé,et al.  Glutathione peroxidase, V. The kinetic mechanism. , 1972, Hoppe-Seyler's Zeitschrift fur physiologische Chemie.

[20]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[21]  C. Little,et al.  An intracellular GSH-peroxidase with a lipid peroxide substrate. , 1968, Biochemical and biophysical research communications.

[22]  R. E. Huber,et al.  Comparison of the chemical properties of selenocysteine and selenocystine with their sulfur analogs. , 1967, Archives of biochemistry and biophysics.

[23]  W. Valentine,et al.  Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. , 1967, The Journal of laboratory and clinical medicine.

[24]  G. C. Mills.  Hemoglobin catabolism. I. Glutathione peroxidase, an erythrocyte enzyme which protects hemoglobin from oxidative breakdown. , 1957, The Journal of biological chemistry.