Active site contribution to specificity of the aspartic proteases plasmepsins I and II.
Plasmepsins I and II (PM I and II) are aspartic proteases involved in the initial steps of Plasmodium hemoglobin degradation. They are attractive targets for antimalarial drug development. The two enzymes are 73% identical, yet have different substrate and inhibitor specificities. The x-ray structures of proform and mature PM II have been determined, but models of PM I do not adequately explain the selectivity of the two proteases. To better understand the basis of these recognition differences, we have identified nine residues of PM II that are in proximity to the inhibitor pepstatin in the crystal structure and differ in PM I. We mutated these residues in PM II to the cognate amino acids of PM I. Kinetic parameters for substrate and inhibitors for the PM II-mutant were similar to those of PM II-wild type (WT). Cleavage specificity was assessed using hemoglobin or a random decamer peptide library as substrate. Again, PM II-mutant behaved like PM II-WT rather than PM I-WT. These results indicate that differences in plasmepsin specificity depend more on conformational differences from distant sites than on specific active site variation.
Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand.
Pilaiwan
P
Siripurkpong
Jirundon
J
Yuvaniyama
Prapon
P
Wilairat
Daniel E
DE
Goldberg
D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D006454:Hemoglobins / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D006454:Hemoglobins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C089643:plasmepsin; C100637:plasmepsin II