Active Site Contribution to Specificity of the Aspartic Proteases Plasmepsins I and II

Siripurkpong, Pilaiwan; Yuvaniyama, Jirundon; Wilairat, Prapon; Goldberg, Daniel E.

doi:10.1074/jbc.m204852200

Cited by 21 publications

(22 citation statements)

References 22 publications

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“…Analyses of substrate preferences by Siripurkpong et al 104 showed an increased preference of Plm I for serine in the P1 0 position, whereas Plm II preferred leucine and methionine in this position. We also found that changes in the P1 0 position of basic hydroxyethylamine inhibitors, related to inhibitors 20-24, resulted in different Plm I/II selectivities, indicating the S1 0 subsite to be significantly different in the two plasmepsins.…”

Section: Hydroxyethylaminesmentioning

confidence: 97%

“…104 Hence, it is evident that remote amino acids from the active site probably play important roles in substrate and inhibitor binding, possibly by inducing different conformations of the active site. To examine which of the plasmepsins, I or II, is most important in parasite growth, several Plm I-and II-selective inhibitors have been assessed in erythrocyte-based assays.…”

Section: B Plasmepsin I Inhibitorsmentioning

confidence: 99%

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Plasmepsins as potential targets for new antimalarial therapy

2006

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Malaria is one of the major diseases in the world. Due to the rapid spread of parasite resistance to available antimalarial drugs there is an urgent need for new antimalarials with novel mechanisms of action. Several promising targets for drug intervention have been revealed in recent years. This review addresses the parasitic aspartic proteases termed plasmepsins (Plms) that are involved in the hemoglobin catabolism that occurs during the erythrocytic stage of the malarial parasite life cycle. Four Plasmodium species are responsible for human malaria; P. vivax, P. ovale, P. malariae, and P. falciparum. This review focuses on inhibitors of the haemoglobin-degrading plasmepsins of the most lethal species, P. falciparum; Plm I, Plm II, Plm IV, and histo-aspartic protease (HAP). Previously, Plm II has attracted the most attention. With the identification and characterization of new plasmepsins and the results from recent plasmepsin knockout studies, it now seems clear that in order to achieve high-antiparasitic activities in P. falciparum-infected erythrocytes it is necessary to inhibit several of the haemoglobin-degrading plasmepsins. Herein we summarize the structure-activity relationships of the Plm I, II, IV, and HAP inhibitors. These inhibitors represent all classes which, to the best of our knowledge, have been disclosed in journal articles to date. The 3D structures of inhibitor/plasmepsin II complexes available in the protein data bank are briefly discussed and compared.

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Section: Hydroxyethylaminesmentioning

confidence: 97%

Section: B Plasmepsin I Inhibitorsmentioning

confidence: 99%

Plasmepsins as potential targets for new antimalarial therapy

2006

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“…The preferences at the primed positions have been characterized in detail with a random decamer peptide library, and it was shown that Leu and Ser are preferred at P1Ј and P2Ј, respectively, whereas there was little specificity at other positions (30). The availability of related sequences has been beneficial to elucidate the substrate specificity of PM II.…”

Section: Discussionmentioning

confidence: 99%

Distal Substrate Interactions Enhance Plasmepsin Activity

Istvan

Goldberg

2005

Journal of Biological Chemistry

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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 sitedirected 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 ␣-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.

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“…Although plasmepsins from each of the four species have been cloned and expressed as recombinant enzymes (12,18,22,44,45), there is still very little known about many of the peptidases recently discovered from the P. falciparum genome-sequencing project (46). The most fully characterized plasmepsin is PfPM2, where numerous structures (18)(19)(20) and kinetic studies (21,31,45,47) have aided in identifying the determinants of substrate specificity.…”

Section: Discussionmentioning

confidence: 99%

Active-Site Specificity of Digestive Aspartic Peptidases from the Four Species of Plasmodium that Infect Humans Using Chromogenic Combinatorial Peptide Libraries

et al. 2005

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Two targeted chromogenic octapeptide combinatorial libraries, comprised of 38 pools each containing 361 different peptides, were used to analyze the enzyme/substrate interactions of five plasmepsins. The first library (P1 library) was based on a good synthetic aspartic peptidase substrate [Westling, J., Cipullo, P., Hung, S. H., Saft, H., Dame, J. B., and Dunn, B. M. (1999) Protein Sci. 8, 2001-2009 Scarborough, P. E., and Dunn, B. M. (1994) Protein Eng. 7, 495-502] and had the sequence Lys-Pro-(Xaa)-Glu-P1*Nph-(Xaa)-Leu. The second library (P1′ library) incorporated results with the plasmepsins from the first library and had the sequence Lys-Pro-Ile-(Xaa)-Nph*P1′-Gln-(Xaa). In both cases, P1 and P1′ were fixed residues for a given peptide pool, where Nph was a para-nitrophenylalanine chromogenic reporter and Xaa was a mixture of 19 different amino acids. Kinetic assays monitoring the rates of cleavage of these libraries revealed the optimal P1 and P1′ residues for the five plasmepsins as hydrophobic substitutions. Extended specificity preferences were obtained utilizing liquid chromatographymass spectrometry (LC-MS) to analyze the cleavage products produced by enzyme-catalyzed digestion of the best pools of each peptide library. LC-MS analysis of the P1-Phe and P1′-Phe pools revealed the favored amino acids at the P3, P2, P2′, and P3′ positions. These analyses have provided new insights on the binding preferences of malarial digestive enzymes that were used to design specific methyleneamino peptidomimetic inhibitors of the plasmepsins. Some of these compounds were potent inhibitors of the five plasmepsins, and their possible binding modes were analyzed by computational methods.

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Active Site Contribution to Specificity of the Aspartic Proteases Plasmepsins I and II

Cited by 21 publications

References 22 publications

Plasmepsins as potential targets for new antimalarial therapy

Plasmepsins as potential targets for new antimalarial therapy

Distal Substrate Interactions Enhance Plasmepsin Activity

Active-Site Specificity of Digestive Aspartic Peptidases from the Four Species of Plasmodium that Infect Humans Using Chromogenic Combinatorial Peptide Libraries

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