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 mut ated to produce a ceruloplasmin stable to proteolysis. The stable cerulopla smin 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 cerul oplasmin were produced in the yeast Pichia pastoris and characterized. The mutations R481A, R701A, and K887A were at the proteolytic sites, did not al ter 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 spectros copic and catalytic properties of ceruloplasmin, supporting the hypothesis that this site is reduced and locked in a rigid frame. In contrast the muta tion C1021S at the type 1 site of domain 6 substantially altered the molecu lar properties of the protein, leaving a small fraction endowed with oxidas e activity. This result, while indicating the importance of this site in st abilizing 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 membrane s of yeast not harboring the ceruloplasmin gene. This indicates that expres sion of ceruloplasmin induces a state of iron deficiency in yeast because t he ferric iron produced in the medium by its ferroxidase activity is not av ailable for the uptake.