Dipeptidyl
aminopeptidase 1 (DPAP1) is an essential food vacuole
enzyme with a putative role in
hemoglobin catabolism by the erythrocytic
malaria parasite. Here, the biochemical properties of DPAP1 have been investigated and compared to those of the human ortholog
cathepsin C. To facilitate the characterization of DPAP1, we have developed a method for the production of purified recombinant DPAP1 with properties closely resembling those of the native
enzyme. Like
cathepsin C, DPAP1 is a
chloride-activated
enzyme that is most efficient in catalyzing
amide bond hydrolysis at acidic pH values. The monomeric quaternary structure of DPAP1 differs from the homotetrameric structure of
cathepsin C, which suggests that tetramerization is required for a
cathepsin C-specific function. The S1 and S2 subsite preferences of DPAP1 and
cathepsin C were profiled with a positional scanning synthetic combinatorial library. The S1 preferences bore close similarity to those of other C1-family
cysteine peptidases. The S2 subsites of both DPAP1 and
cathepsin C accepted aliphatic hydrophobic residues,
proline, and some polar residues, yielding a distinct specificity profile. DPAP1 efficiently catalyzed the hydrolysis of several fluorogenic
dipeptide substrates; surprisingly, however, a potential substrate with a P2-phenylalanine residue was instead a competitive inhibitor. Together, our biochemical data suggest that DPAP1 accelerates the production of
amino acids from
hemoglobin by bridging the gap between the
endopeptidase and
aminopeptidase activities of the food vacuole. Two reversible
cathepsin C inhibitors potently inhibited both recombinant and native DPAP1, thereby validating the use of recombinant DPAP1 for future inhibitor discovery and characterization.