title: Loss of in vitro metal ion binding specificity in mutant copper-zinc superoxide dismutases associated with familial amyotrophic lateral sclerosis.
abstract: The presence of the copper ion at the active site of human wild type copper-zinc superoxide dismutase (CuZnSOD) is essential to its ability to catalyze the disproportionation of superoxide into dioxygen and hydrogen peroxide. Wild type CuZnSOD and several of the mutants associated with familial amyotrophic lateral sclerosis (FALS) (Ala(4) --> Val, Gly(93) --> Ala, and Leu(38) --> Val) were expressed in Saccharomyces cerevisiae. Purified metal-free (apoproteins) and various remetallated derivatives were analyzed by metal titrations monitored by UV-visible spectroscopy, histidine modification studies using diethylpyrocarbonate, and enzymatic activity measurements using pulse radiolysis. From these studies it was concluded that the FALS mutant CuZnSOD apoproteins, in direct contrast to the human wild type apoprotein, have lost their ability to partition and bind copper and zinc ions in their proper locations in vitro. Similar studies of the wild type and FALS mutant CuZnSOD holoenzymes in the "as isolated" metallation state showed abnormally low copper-to-zinc ratios, although all of the copper acquired was located at the native copper binding sites. Thus, the copper ions are properly directed to their native binding sites in vivo, presumably as a result of the action of the yeast copper chaperone Lys7p (yeast CCS). The loss of metal ion binding specificity of FALS mutant CuZnSODs in vitro may be related to their role in ALS.
authors.affiliation: Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095-1569, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Goto
authors.forename: H
authors.initials: H
authors.lastname: Zhu
authors.forename: R J
authors.initials: RJ
authors.lastname: Sanchez
authors.forename: A
authors.initials: A
authors.lastname: Nersissian
authors.forename: E B
authors.initials: EB
authors.lastname: Gralla
authors.forename: J S
authors.initials: JS
authors.lastname: Valentine
authors.forename: D E
authors.initials: DE
authors.lastname: Cabelli
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D003300:Copper / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D016472:Motor Neuron Disease / Q000201:enzymology* / Q000235:genetics*; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013053:Spectrophotometry; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D003300:Copper; D013482:Superoxide Dismutase