Site-directed mutagenesis of human ceruloplasmin:. production of a proteolytically stable protein and structure-activity relationships of type 1 sites.
A fully active recombinant human ceruloplasmin was obtained, and it was mutated to produce a ceruloplasmin stable to proteolysis. The stable ceruloplasmin was further mutated to perturb the environment of copper at the type 1 copper sites in two different domains. The wild type and the mutated ceruloplasmin were produced in the yeast Pichia pastoris and characterized. The mutations R481A, R701A, and K887A were at the proteolytic sites, did not alter the enzymatic activity, and were all necessary to protect ceruloplasmin from degradation. The mutation L329M was at the tricoordinate type 1 site of the domain 2 and was ineffective to induce modifications of the spectroscopic and catalytic properties of ceruloplasmin, supporting the hypothesis that this site is reduced and locked in a rigid frame. In contrast the mutation C1021S at the type 1 site of domain 6 substantially altered the molecular properties of the protein, leaving a small fraction endowed with oxidase activity. This result, while indicating the importance of this site in stabilizing the overall protein structure, suggests that another type 1 site is competent for dioxygen reduction. During the expression of ceruloplasmin, the yeast maintained a high level of Fet3 that was released from membranes of yeast not harboring the ceruloplasmin gene. This indicates that expression of ceruloplasmin induces a state of iron deficiency in yeast because the ferric iron produced in the medium by its ferroxidase activity is not available for the uptake.
Bielli
P
P
Department of Biology, University Roma Tre, Rome, Italy. pamela@gem.univie.ac.at
Bellenchi
G C
GC
Calabrese
L
L
D000595:Amino Acid Sequence; D002570:Ceruloplasmin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003300:Copper; D010450:Endopeptidases / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010843:Pichia / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D003300:Copper; D002570:Ceruloplasmin; C085513:FET3 protein, S cerevisiae; D010450:Endopeptidases