title: Copper-dependent inhibition and oxidative inactivation with affinity cleavage of yeast glutathione reductase.
abstract: Effects of copper on the activity and oxidative inactivation of yeast glutathione reductase were analyzed. Glutathione reductase from yeast was inhibited by cupric ion and more potently by cuprous ion. Copper ion inhibited the enzyme noncompetitively with respect to the substrate GSSG and NADPH. The Ki values of the enzyme for Cu(2+) and Cu(+) ion were determined to be 1 and 0.35 μM, respectively. Copper-dependent inactivation of glutathione reductase was also analyzed. Hydrogen peroxide and copper/ascorbate also caused an inactivation with the cleavage of peptide bond of the enzyme. The inactivation/fragmentation of the enzyme was prevented by addition of catalase, suggesting that hydroxyl radical produced through the cuprous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF-MS analysis confirmed eight fragments, which were further determined to result from the cleavage of the Met17-Ser18, Asn20-Thr21, Glu251-Gly252, Ser420-Pro421, Pro421-Thr422 bonds of the enzyme by amino-terminal sequencing analysis. Based on the kinetic analysis and no protective effect of the substrates, GSSG and NADPH on the copper-mediated inactivation/fragmentation of the enzyme, copper binds to the sites apart from the substrate-sites, causing the peptide cleavage by hydroxyl radical. Copper-dependent oxidative inactivation/fragmentation of glutathione reductase can explain the prooxidant properties of copper under the in vivo conditions.
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Yazako-Karimata 1-1, Nagakute, Aichi, 480-1195, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Murakami
authors.forename: Ryoko
authors.initials: R
authors.lastname: Tsubouchi
authors.forename: Minoru
authors.initials: M
authors.lastname: Fukayama
authors.forename: Masataka
authors.initials: M
authors.lastname: Yoshino
mesh_terms: D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000737:chemistry; D019327:Copper Sulfate / Q000737:chemistry*; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D017665:Hydroxyl Radical / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*
chemical_list: D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D017665:Hydroxyl Radical; D005980:Glutathione Reductase; D019327:Copper Sulfate; D001205:Ascorbic Acid