Distal substrate interactions enhance plasmepsin activity.
Plasmepsin II (PM II) is an aspartic protease active in hemoglobin (Hb) degradation in the protozoan parasite Plasmodium falciparum. A fluorescence-quenched octapeptide substrate based on the initial hemoglobin cleavage site is recognized well by PM II. C-terminal extension of this peptide has little effect, but N-terminal extension results in higher maximal velocity and dramatic concentration-dependent substrate inhibition. This inhibition, but not the rate stimulation, depends on the presence of a DABCYL group on the peptide N terminus. Using site-directed mutagenesis, we have identified PM II residues that interact with N-terminal amino acids of peptide substrates. The same residues influence degradation of Hb by PM II. Cathepsin E (CatE), a related mammalian aspartic protease, is also stimulated by N-terminally extended substrates. This suggests that distal substrate interactions as far out as P6 may be a general property of aspartic proteases and may be important in substrate and inhibitor recognition. Although PM II and CatE are similar in their ability to cleave Hb-based peptides and Hb alpha-chains, CatE is not able to degrade native Hb, which is a substrate for PM II. Based on these results, we propose that PM II may have the special feature of being a Hb denaturase.
Istvan
Eva S
ES
Department of Medicine, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Goldberg
Daniel E
DE
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000201:enzymology*; D015800:Protozoan Proteins; D013379:Substrate Specificity
D006454:Hemoglobins; D010446:Peptide Fragments; D015800:Protozoan Proteins; D016282:Aspartic Acid Endopeptidases; C100637:plasmepsin II