Analysis of the S-2 subsite specificities of the recombinant cysteine proteinases CPB of Leishmania mexicana, and cruzain of Trypanosoma cruzi, usingfluorescent substrates containing non-natural basic amino acids

We have explored the specificity of the S-2 subsite of recombinant cysteine proteinases from Leishmania mexicana (CPB2.8 Delta CTE) and from Trypanoso ma cruzi (cruzain) employing a series of fluorogenic substrates based on th e peptide Bz-F-R-MCA, in which Bz is the benzoyl group and the Phe residue has been substituted for by Arg, His and non-natural basic amino acids that combine a basic group with an aromatic or hydrophobic group at the side ch ain: 4-aminomethyl-phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4- aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-pip eridinylalanine (Ppa). 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminoc yclohexyl-alanine (Aca). Bz-F-R-MCA was hydrolyzed well by CPB2.8 Delta CTE and cruzain, but all the substitutions of Phe resulted in less susceptible substrates for the two enzymes. CPB2.8 Delta CTE has a restricted specific ity to hydrophobic side chains as with cathepsin L. However, the peptides w ith the residues Amf and Ama presented higher affinity to CPB2.8ACTE, and t he latter was an inhibitor of the enzyme. Although, cruzain accepts basic a s well as hydrophobic residues at the S2 Subsite, it is more restrictive th an cathepsin B and no inhibitor was found amongst the examined peptides. (C ) 2001 Elsevier Science B.V. All rights reserved.