Falcipain-2 is one of the principal hemoglobinases of Plasmoodium falciparum, a human malaria parasite. It has a typical papain family cysteine protease structural organization, a large pro-domain, a mature domain with conserved active site amino acids. Prodomain of falcipain-2 also contains two important conserved motifs, "GNFD" and "ERFNIN." The "GNFD" motif has been shown to be responsible for correct folding and stability in case of many papain family proteases. In the present study, we carried out site-directed mutagenesis to assess the roles of active site residues and pro-domain residues for the activity of 1 falcipain-2. Our results showed that substitutions of putative active site residues; Q36, C42 H174, and N204 resulted in complete loss of falcipain-2 activity, while W206 and D155 mutants retained partial/complete activity in comparison to the wild type falcipain-2. Homology modeling data also corroborate the results of mutagenesis Q36, C42, H 1 74 N204, and W206 residues form the active site loop of the enzyme and D155 lie outside the active pocket. Substitutions in the pro-region did not affect the activity of falcipain-2. This implies that falcipain-2 shares active site residues with other members of papain family, however pro-region of falcipain-2 does not play any role in the activity of enzyme. (C) 2004 Elsevier Inc. All rights reserved.