title: Homology modeling and mutagenesis analyses of Plasmodium falciparum falcipain 2A: implications for rational drug design.
abstract: The hemoglobin-degrading cysteine proteases falcipains of the malaria parasite Plasmodium falciparum are regarded as potential drug targets. Despite their obvious importance in the virulence of malaria, these proteases remain poorly characterized at the structural levels. Using a bioinformatic and site-directed mutagenesis approach, residues essential for the structure and function of FP2A are elucidated in this study. In total, nine mutants of FP2A were constructed to test the proposed importance of seven different amino acid residues. These recombinant protease mutants were solubly expressed in Escherichia coli and purified by affinity chromatography for enzymatic assessments. Notably, substitutions at positions C99 and C119 induce structural alterations and led to significant reduction in enzyme activity (>97%). The analyses also validated the role of the active triad comprising of C42, H174, and N204 in catalysis and identified a serine at position 149 which is required for specific peptide substrate interactions. The parasite-specific residues, C99, C119, and S149, represent potential sites for differential targeting, since the corresponding residues are absent in the human host's isozymes.
authors.affiliation: Department of Microbiology, Faculty of Medicine, National University of Singapore, MD4A, 5 Science Drive 2, Singapore 117597, Singapore.
authors.forename: Liuh Ling
authors.initials: LL
authors.lastname: Goh
authors.forename: Tiow Suan
authors.initials: TS
authors.lastname: Sim
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003198:Computer Simulation; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D016503:Drug Delivery Systems / Q000379:methods; D015195:Drug Design; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C416224:falcipain 2