Identification of catalytically important amino acids in human ceruloplasmin by site-directed mutagenesis.
The involvement of amino acid residues previously proposed on the basis of structural data to have roles in the ferroxidase and diamine oxidase activities of human ceruloplasmin was investigated. Variants of human ceruloplasmin, in which residues proposed to be involved in electron transfer and/or iron-binding had been altered by site-directed mutagenesis, were expressed in HEK293 cells. E633A and E597A/H602A variants exhibited reduction in both activities by 50-60% compared to recombinant wild-type ceruloplasmin. The variant E935A/H940A had reduced ferroxidase activity (50%) but unaltered diamine oxidase activity, whereas the variant E971A exhibited enhanced diamine oxidase activity. For the L329M variant, both activities were identical to those of wild-type ceruloplasmin.
Brown
Mark A
MA
Department of Biochemistry and Molecular Biology, and the UBC Centre for Blood Research, Faculty of Medicine, University of British Columbia, 2146 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3. mark.brown@klkemi.mas.lu.se
Stenberg
Leisa M
LM
Mauk
A Grant
AG
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics*; D015153:Blotting, Western; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D002570:Ceruloplasmin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D000596:Amino Acids; D011994:Recombinant Proteins; D002570:Ceruloplasmin