Biochemical characterization of aminopeptidase N2 from Toxoplasmagondii

[摘要] Aminopeptidase N (APN) is a member of the highly conserved M1 family of metalloproteases,and is considered to be a valuable target for the treatment of a variety of diseases,e.g., cancer, malaria, and coccidiosis. In this study, we identified an APN gene(TgAPN2) in the Toxoplasma gondii genome, andperformed a biochemical characterization of the recombinant TgAPN2(rTgAPN2) protein. Active rTgAPN2 was first producedand purified in Escherichia coli. The catalytic activity of the enzymewas verified using a specific fluorescent substrate, H-Ala-MCA; therTgAPN2 was relatively active in the absence of added metal ions. Theaddition of some metal ions, especially Zn2+, inhibited the activity of therecombinant enzyme. The activity of rTgAPN2 was reduced in the presenceof the EDTA chelator in the absence of added metal ions. The optimum pH for enzymeactivity was 8.0; the enzyme was active in the 3–10 pH range. The substrate preference ofrTgAPN2 was evaluated. The enzyme showed a preference for substratescontaining N-terminal Ala and Arg residues. Finally, bestatin and amastatin were shown toinhibit the activity of the enzyme. In conclusion, rTgAPN2 shared generalcharacteristics with the M1 family of aminopeptidases but also had some uniquecharacteristics. This provides a basis for the function of aminopeptidases and the studyof drug targets.

[学科分类] 兽医学

[关键词] activity;aminopeptidase N (APN);enzyme;Toxoplasma gondii