INVESTIGATION OF THE SUBSTRATE-SPECIFICITY OF CRUZIPAIN, THE MAJOR CYSTEINE PROTEINASE OF TRYPANOSOMA-CRUZI, THROUGH THE USE OF CYSTATIN-DERIVED SUBSTRATES AND INHIBITORS

A panel of intramolecularly quenched fluorogenic substrates containing the conserved QVVA and LVG inhibitory sequences of cystatin inhibitor s was used to describe the specificity of the major cysteine proteinas e of Trypanosoma cruzi (cruzipain or cruzain). This approach was based on the observations that: (1) cruzipain is strongly inhibited by chic ken cystatin and rat T-kininogen, two representative members of cystat in families 2 and 3; (2) the QVVA- and LVG-containing substrates are s pecifically hydrolysed by papain-like proteinases; and (3) the cystati n-like motifs arl similar to the proteolytically sensitive sequences i n cruzipain that separate the pro-region and/or the C-terminal extensi on from the catalytic domain. Specificity constants (k(cat.)/K-m) were determined and compared with those of mammalian cathepsins B and L fr om rat liver lysosomes. Cruzipain and the mammalian proteinases cleave d cystatin-derived substrates at the same site, but their specificitie s differed significantly. Increased specificity for cruzipain was obta ined by replacing amino acids at critical positions on both sides of t he cleavage sites, especially at position P2'. The specificity constan ts (k(cat.)/K-m) obtained for the two substrates with a prolyl residue at P2' (O-aminobenzoyl-QVVAGP-ethlylenediamine 2-4-dinitrophenyl and O-aminobenzoyl-VVGGP-ethylenediamine 2-4-dinitrophenyl) were about 50 times higher for cruzipain than for rat cathepsin L and about 100 time s higher than for cathepsin B. Diazomethylketone derivatives, based on the non-prime sequence of cystatin-derived substrates, inhibited cruz ipain irreversibly, but their inactivation rate constants were conside rably lower than those for mammalian cathepsins B and L, confirming th e importance of P' residues for cruzipain specificity.