Characterization of the Monomer-Dimer Equilibrium of Recombinant Histo-aspartic Protease from Plasmodium falciparum

Abstract

Malaria is a devastating disease that infects and kills 1-2 million people annually. Histo-aspartic protease (HAP) from Plasmodium falciparum, the most lethal of all Plasmodium parasites, is an intriguing aspartic protease due to its unique structure and its potential as an antimalarial target. Substantial effort has been devoted to investigate the structure function of this protease. The present study investigated the molecular state of HAP as related to enzymatic activity. Gel filtration chromatography indicated that recombinant Trx-tHAP fusion protein aggregated during purification and that aggregation could be prevented through the addition of 0.2% CHAPS. Using this latter technique as well as sedimentation velocity and sedimentation equilibrium ultracentrifugation, it was shown that the recombinant mature HAP (mtHAP), in which the His-tag, thioredoxin and prosegement were removed, exists in a dynamic monomer-dimer equilibrium in solution and the dissociation constant is 20-30 μM. Enzymatic activity data also indicated that HAP was most active as a monomer. The monomeric form of mtHAP showed a Km of 9.7 μM and a turnover number, kcat, of 0.044s-1 on the internally quenched fluorescent synthetic peptide substrate EDANS-CO-CH2-CH2-CO-Ala-Leu-Glu-Arg-Met-Phe-Leu-Ser-Phe-Pro-Dap-(DABCYL)-OH (2837b) at pH 6.5. Inhibition studies showed that the activity of mtHAP was completely inhibited by 1 mM PMSF and to a lesser degree by 10 μM ALLN, 10 mM EDTA and 10 mM 1,10-phenanthroline, and was inhibited strongly by ZnCl2 and to a lesser extent by NaCl and KBr. The effects of temperature and salts on the monomer-dimer equilibrium of mtHAP were also investigated by using sedimentation equilibrium ultracentrifugation.