Details

Autor(en) / Beteiligte

• Kolla, Venkata Karunakar
• Prasad, Rajesh
• Sayyad, Zuberwasim
• Atul
• Shah, Akruti Yatendra
• Allanki, Aparna Devi
• Navale, Rahul
• Singhal, Neha
• Tanneru, Nandita
• Sudhakar, Renu
• Venkatesan, Vijayalakshmi
• Deshmukh, Mandar V.
• Sijwali, Puran Singh

Titel

Independent amino acid residues in the S2 pocket of falcipain-3 determine its specificity for P2 residues in substrates

Ist Teil von

• Molecular and biochemical parasitology, 2015-08, Vol.202 (2), p.11-22

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2015

Quelle

Elsevier ScienceDirect Journals

Beschreibungen/Notizen

• [Display omitted] •The S2 pocket residue I94 is chiefly responsible for P2 Leu preference of FP3.•P181 in the S2 pocket renders FP3 negligibly active against Z-RR-AMC.•E243 in FP3 and D234 in FP2 are critical for Z-RR-AMC hydrolysis.•Multiple S2 pocket residues contribute to haemoglobinase activity.•P181 and E243 contribute to the optimum activity of FP3 in the food vacuole pH. Falcipain-3 (FP3) is an essential and drug target cysteine protease of the most lethal human malaria parasite Plasmodium falciparum. FP3 and its majority of homologs in malaria parasites prefer Leu at the P2 position in substrates and inhibitors, whereas its major host homolog cathepsin L prefers Phe. However, FP3 is much less active on peptide substrates and has negligible activity against a P2 Arg-containing substrate (Z-RR-AMC) compared to its paralog falcipain-2A (FP2A). To identify the specificity determinants, the S2/3 pocket residues of FP3 were substituted with the corresponding residues in FP2 or cathepsin L, and the wild type and mutant proteases were assessed for hydrolysis of peptide and protein substrates. Our results indicate that the S2 pocket residues I94 and P181 of FP3 are chiefly responsible for its P2 Leu preference and negligible activity for Z-RR-AMC, respectively. E243 in FP3 and the corresponding residue D234 in FP2 have a key role in Z-RR-AMC hydrolysing activity, possibly through stabilization of side chain interactions, as their substitution with Ala abolished the activity. Several FP3 mutants, which retained P2 Leu preference and showed similar or more activity than wild type FP3 on peptide substrates, degraded haemoglobin less efficiently than wild type FP3, suggesting that multiple residues contribute to haemoglobinase activity. Furthermore, P181 and E243 appear to contribute to the optimum activity of FP3 in the food vacuole milieu (≈pH 5.5). The identification of residues determining specificity of FP3 could aid in developing specific inhibitors of FP3 and its homologs in malaria parasites.