Analysis of the S2 subsite specificities of the recombinant cysteine proteinases CPB of Leishmania mexicana, and cruzain of Trypanosoma cruzi, using fluorescent substrates containing non-natural basic amino acids

Carboxydipeptidase activities of recombinant cysteine peptidases

“…The specificity constant (k cat /K m ) values for the carboxydipeptidase activity of cruzain on the peptides shown in Table 1 are very close to those of cathepsin B and all are higher than the k cat /K m values obtained for cathepsin L. The k cat /K m values of hydrolysis of the peptides Abz-FRFK*-OH, Abz-RRFK*-OH and Abz-ARFK*-OH by cruzain, demonstrated the preference of the S 2 enzyme subsite for a hydrophobic amino acid, which is similar to that observed for its endopeptidase activity [19][20][21][22][23]. It is noteworthy that the k cat /K m value of hydrolysis of Abz-RRFK*-OH by cruzain is lower than that of Abz-FRFK*-OH exclusively by the k cat decrease; the K m value is significantly lower for the hydrolysis of the peptide with Arg at P 2 position.…”

“…As for L. mexicana cysteine peptidases, cruzipain seems to be central to the survival of the parasites in a mammalian host [5,15,16], although the precise mechanisms involved are yet to be fully elucidated [17]. The detailed substrate specificity was earlier reported for cruzipain, the enzyme form isolated from the parasite, and for cruzain, which is the recombinant form without the 100 amino acid C‐terminal extension [18–23]. CPB2.8ΔCTE is a recombinant form of CPB from L. mexicana , again lacking the C‐terminal extension, which has been extensively studied and for which many details of its substrate specificity are available [22–27].…”

“…The detailed substrate specificity was earlier reported for cruzipain, the enzyme form isolated from the parasite, and for cruzain, which is the recombinant form without the 100 amino acid C‐terminal extension [18–23]. CPB2.8ΔCTE is a recombinant form of CPB from L. mexicana , again lacking the C‐terminal extension, which has been extensively studied and for which many details of its substrate specificity are available [22–27]. Cruzain and CPB2.8ΔCTE work as endopeptidases on specific substrates of different sizes, with substantially similar substrate specificity to cathepsin L.…”

Cruzain inhibition by hydroxymethylnitrofurazone and nitrofurazone: investigation of a new target inTrypanosoma cruzi

“…Then, 5 L of the substrate (Bz-Phe-Arg-AMC) stock solution (170 M) was added and the activity of the enzyme was determined through fluorimetric measurements on a Hitachi F4500 spectrofluorimeter (excitation: 380 nm; emission: 460 nm) at 37°C, and under magnetic stirring 15 . The IC 50 of enzymatic inhibition was calculated using the program GraFit 5.0 25 .…”

“…T. cruzi's primary cysteine protease, cruzain or cruzipain, is essential for the infection of host cells, replication, and metabolism throughout the life-cycle of the parasite 12 . This enzyme exhibits a three-dimensional structure similar to those of the papain family proteases such as cathepsin L, but the substrate specificity is reminiscent of cathepsin B, because the S 2 subsite of cruzain accepts basic amino acids quite well [13][14][15] . The search for new nonpeptidic cruzain inhibitors has been used for the development of antichagasic chemotherapy 16 .…”