Exploring the role of putative active site amino acids and pro-region motif of recombinant falcipain-2: a principal hemoglobinase of Plasmodium falciparum.
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.
Kumar
Amit
A
International Center for Genetic Engineering and Biotechnology, PO Box No 10504, Aruna Asaf Ali Marg, New Delhi-110067, India.
Dasaradhi
P V N
PV
Chauhan
Virander S
VS
Malhotra
Pawan
P
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D006454:Hemoglobins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D020673:Protein Renaturation; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D006454:Hemoglobins; D009842:Oligopeptides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C416224:falcipain 2