Characterization of native falcipain, an enzyme involved in Plasmodium falciparum hemoglobin degradation.
In Plasmodium falciparum, a cysteine protease known as falcipain has been implicated in the essential metabolic process of hemoglobin degradation. Parallel lines of investigation, using native or recombinant enzyme, have led to differing conclusions about the specificity and role of this protease. We have now determined that (1) Native falcipain does not cleave hemoglobin unless this substrate has first been denatured by reducing agents, acid-acetone treatment or plasmepsin action. (2) Reducing agents such as glutathione cannot denature hemoglobin in the presence of catalase, which is accumulated in the digestive vacuole. (3) The purified native enzyme has kinetics similar to those obtained with trophozoite extract, but substantially different from those of recombinant enzyme. (4) Although there are numerous cysteine protease genes in the P. falciparum genome, the falcipain gene is the only one whose transcript can be detected in the early intraerythrocytic parasites. We conclude that falcipain likely works by degrading hemoglobin fragments after initial aspartic protease attack has denatured the substrate. We propose that falcipain inhibitors block the initial steps of degradation indirectly by promoting vacuolar accumulation of osmotically active hemoglobin peptides.
Francis
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SE
Howard Hughes Medical Institute, Department of Molecular Microbiology, St. Louis, Missouri 63110, USA.
Gluzman
I Y
IY
Oksman
A
A
Banerjee
D
D
Goldberg
D E
DE
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D016054:DNA, Protozoan / Q000032:analysis; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010963:Plasmodium falciparum / Q000201:enzymology*; D011489:Protein Denaturation; D016053:RNA, Protozoan / Q000032:analysis; D019163:Reducing Agents / Q000494:pharmacology; D014617:Vacuoles / Q000201:enzymology
D015853:Cysteine Proteinase Inhibitors; D016054:DNA, Protozoan; D006454:Hemoglobins; D016053:RNA, Protozoan; D019163:Reducing Agents; D002374:Catalase; D003546:Cysteine Endopeptidases; C099607:falcipain; D016282:Aspartic Acid Endopeptidases; C089643:plasmepsin; D007930:Leucine; C024974:E 64