New hyperthermostable aminopeptidase from the hyperthermophilic archaeon Pyrococcus horikoshii has acylamino acid releasing (deblocking) activity for acyl (blocked) peptides. Such an enzyme can be used for N-terminal sequencing of acyl peptides. To clarify the active site of the deblocking aminopeptidase, we prepared three mutants in which one of the three possible active site amino acid residues (Asp or Glu) was replaced with their amide derivatives. Activity and cobalt ion dependence of these mutants were examined and compared with those of the native enzyme. The results suggest that all the three possible residues (Asp173, Glu205, and Glu206) participate in the catalytic activity through binding with the cobalt ion. (C) 2002 Elsevier Science (USA).