Falcipain-2 is one of the principal hemoglobinases of Plasmodium 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. Pro-domain 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
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, H174, 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.