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Journal of Inorganic Biochemistry
Lo, SY;Sbel, CE;Webb, MI;Walsby, CJ;Siemann, S;
Anthrax lethal factor (LF) is a zinc-dependent metalloendopeptidase and a member of the gluzincin family. The current report demonstrates a high metal substitution tolerance of LF atypical of gluzincins and other zinc-dependent metalloproteases. Mn(2+), Co(2+), Ni(2+), Cu(2+) and Cd(2+) were found to reactivate the apoprotein of LF to a level either comparable to or significantly higher than that noted for the native zinc enzyme. The most active form of LF was obtained with Cu(2+), a surprising observation since most Cu(2+)-substituted zinc proteases display very low activity. Cu(2+)-substituted LF (CuLF), prepared by direct exchange and by apoprotein reconstitution methodologies, displayed a several-fold higher catalytic competence towards chromogenic and fluorogenic LF substrates than native LF. CuLF bound Cu(2+) tightly with a dissociation constant in the femtomolar range. The electron paramagnetic resonance spectrum of CuLF revealed the protein-bound metal ion to be coordinated to two nitrogen donor atoms, suggesting that Cu(2+) binds to both active site histidine residues. While ZnLF and CuLF (prepared by direct exchange) were capable of killing RAW 264.7 murine macrophage-like cells, apoLF and all metal-reconstituted apoprotein preparations failed to elicit a cytotoxic response. Competition experiments using apoLF/ZnLF mixtures demonstrated the propensity of apoLF to relieve ZnLF-induced cell death, suggesting that both protein forms can compete with each other for binding to protective antigen. The lack of cytotoxicity of apoLF and its metal-reconstituted variants likely originates from structural perturbations in these proteins which might prevent their translocation into the cytoplasm.