Carboxydipeptidase activities of recombinant cysteine peptidases. Cruzain of Trypanosoma cruzi and CPB of Leishmania mexicana.
The recombinant cysteine peptidases, cruzain from Trypanosoma cruzi and CPB2.8DeltaCTE from Leishmania mexicana, are cathepsin L-like and characteristically endopeptidases. In this study, we characterized the carboxydipeptidase activities of these enzymes and compared them with those of human recombinant cathepsin B and cathepsin L. The analysis used the internally quenched fluorescent peptide Abz-FRFK*-OH and some of its analogues, where Abz is ortho-aminobenzoic acid and K* is (2,4-dinitrophenyl)-epsilon-NH2-lysine. These peptides were demonstrated to be very sensitive substrates, due to the strong quenching effect of K* on the fluorescence of the Abz group. The carboxydipeptidase activity of cruzain was shown to be very similar to that of cathepsin B, while that of CPB2.8DeltaCTE is closer to the carboxydipeptidase activity of cathepsin L. The S2 subsite architecture of cruzain and the nature of the amino acid at the P2 position of the substrates determine its carboxydipeptidase activity and gives further and direct support to the notion that the carboxydipeptidase activity of the papain family cysteine peptidases rely on the S2-P2 interaction [Nägler D. K., Tam, W., Storer, A.C., Krupa, J.C., Mort, J.S. & Menard, R. (1999) Biochemistry38, 4868-4874]. Cruzain and CPB2.8DeltaCTE presented a broad pH-range for both the endo- and exo-peptidase activities, although the later is approximately one order of magnitude lower. This feature, that is not common in related mammalian cysteine peptidases, is consistent with the enzymes being exposed to different environmental conditions and having different locations during parasite development.
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, 04044-20 São Paulo, Brazil.
Wagner A S
WA
Judice
Luciano
L
Puzer
Simone S
SS
Cotrin
Adriana K
AK
Carmona
Graham H
GH
Coombs
Luiz
L
Juliano
Maria A
MA
Juliano
D000818:Animals; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism*; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007894:Leishmania mexicana / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes; D010455:Peptides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D002268:Carboxypeptidases; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C074032:cruzain, Trypanosoma cruzi; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L