Plasmodium falciparum:: Biochemical characterization of the cysteine protease falcipain-2′

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 are hemoglobinases and potential antimalarial drug targets. The falcipain-2' gene was identified recently and is nearly identical in sequence to falcipain-2. The product of this gene has not been studied previously. The mature protease domain of falcipain-2' was expressed in Escherichia coli, purified, and refolded to active enzyme. Functional analysis revealed similar biochemical properties to those of falcipain-2, including pH optima (pH 5.5-7.0), reducing requirements, and substrate preference. Studies with cysteine protease inhibitors showed similar inhibition of falcipain-2 and falcipain-2', although specificities were not identical. Considering activity against the presumed biological substrate, both enzymes readily hydrolyzed hemoglobin. Our results confirm that falcipain-2' is an active hemoglobinase and suggest that falcipain-2 and falclpain-2' play similar roles in erythrocytic parasites but that, for promising cysteine protease inhibitors, it will be important to confirm activity against this additional target. (c) 2005 Elsevier Inc. All rights reserved.