Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target.
Bottom Line: Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif.Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates.Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease.
Affiliation: Division of Parasitology, MRC National Institute for Medical Research (NIMR), Mill Hill, London NW7 1AA, UK.Show MeSH
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Mentions: To address the potential importance of the P1′ Ser residue in substrate cleavage by PbSUB1, we explored whether a PfSUB1 substrate that is a poor substrate for PbSUB1 could be converted to a better PbSUB1 substrate by substitution of the P1′ residue with Ser. We produced the fluorogenic substrate SERA4st1F-6R, which is the peptide Ac-CITAQDDEEC (corresponding to P4–P4′ of the SERA4st1 cleavage site) labelled on both terminal cysteine side-chains with 6-IATR. In parallel, we generated the derivative SERA4st1F-6R-S, which contains a Ser substitution of the P1′ (Asp) residue in SERA4st1F-6R. Cleavage of SERA4st1F-6R by rPfSUB1cat resulted in the expected florescence increase, with conversion of the substrate to just two fluorescent products corresponding to cleavage at the expected Gln-Asp bond, as determined by RP-HPLC and mass-spectrometry (data not shown). Fig. 8 shows a kinetic analysis comparing cleavage of SERA4st1F-6R and SERA4st1F-6R-S by rPfSUB1cat and rPbSUB1cat. At a standard concentration of both substrates (0.1 μM) which is well below their Km and therefore corresponding to conditions where the rate of cleavage is directly proportional to kcat/Km, it can be seen that whereas rPfSUB1cat cleaves both substrates equally well, SERA4st1F-6R-S is a much better substrate for rPbSUB1cat than SERA4st1F-6R. These results confirm that – as in the case of PfSUB1 – prime side resides do indeed contribute to substrate recognition by PbSUB1, and suggest that PbSUB1 prefers P1′ Ser to Asp, whereas PfSUB1 does not discriminate between them under these conditions. The observation that authentic PbSUB1 cleavage sites in at least two essential proteins possess P1′ Ser suggests that the enzyme has co-evolved with its cognate endogenous substrates in order to maintain optimal catalytic activity.
Affiliation: Division of Parasitology, MRC National Institute for Medical Research (NIMR), Mill Hill, London NW7 1AA, UK.