title: Knockout of SOD1 promotes conversion of selenocysteine to dehydroalanine in murine hepatic GPX1 protein.
abstract: Se-dependent glutathione peroxidase-1 (GPX1) and Cu,Zn-superoxide dismutase (SOD1) are two major intracellular antioxidant enzymes. The purpose of this study was to elucidate the biochemical mechanisms for the 40% loss of hepatic GPX1 activity in SOD1(-/-) mice. Compared with the wild type (WT), the SOD1(-/-) mice showed no change in the total amount of GPX1 protein. However, their total enzyme protein exhibited 31 and 38% decreases (P<0.05) in the apparent k(cat) for hydrogen peroxide and tert-butylperoxide (at 2mM GSH), respectively. Most striking, mass spectrometry revealed two chemical forms of the 47th residue of GPX1: the projected native selenocysteine (Sec) and the Se-lacking dehydroalanine (DHA). The hepatic GPX1 protein of the SOD1(-/-) mice contained 38% less Sec and 77% more DHA than that of WT and showed aggravated dissociation of the tetramer structure. In conclusion, knockout of SOD1 elevated the conversion of Sec to DHA in the active site of hepatic GPX1, leading to proportional decreases in the apparent k(cat) and activity of the enzyme protein as a whole. Our data reveal a structural and kinetic mechanism for the in vivo functional dependence of GPX1 on SOD1 in mammals and provide a novel mass spectrometric method for the assay of oxidative modification of the GPX1 protein.
authors.affiliation: Department of Animal Science, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Shi Kui
authors.initials: SK
authors.lastname: Wang
authors.forename: Jeremy D
authors.initials: JD
authors.lastname: Weaver
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhang
authors.forename: Xin Gen
authors.initials: XG
authors.lastname: Lei
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005838:Genotype; D005979:Glutathione Peroxidase / Q000737:chemistry / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017279:Selenocysteine / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000172:deficiency / Q000378:metabolism*; D000072105:Superoxide Dismutase-1; D020122:tert-Butylhydroperoxide / Q000378:metabolism; D000094765:Glutathione Peroxidase GPX1
chemical_list: D017279:Selenocysteine; D020122:tert-Butylhydroperoxide; C015102:dehydroalanine; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D000409:Alanine; D000094765:Glutathione Peroxidase GPX1; C000722068:Gpx1 protein, mouse