S-1 subsite specificity of a recombinant cysteine proteinase, CPB, of Leishmania mexicana compared with cruzain, human cathepsin L and papain using substrates containing non-natural basic amino acids

S-1 subsite specificity of a recombinant cysteine proteinase, CPB, of Leishmania mexicana compared with cruzain, human cathepsin L and papain using substrates containing non-natural basic amino acids

Autor Alves, Lira C. Autor UNIFESP Google Scholar
Melo, Robson Lopes de Autor UNIFESP Google Scholar
Sanderson, S. J. Google Scholar
Mottram, J. C. Google Scholar
Coombs, G. H. Google Scholar
Caliendo, G. Google Scholar
Santagada, V Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Instituição Universidade Federal de São Paulo (UNIFESP)
Univ Glasgow
Univ Naples
Resumo We have explored the substrate specificity of a recombinant cysteine proteinase of Leishmania mexicana (CPB2.8 Delta CTE) in order to obtain data that will enable us to design specific inhibitors of the enzyme. Previously we have shown that the enzyme has high activity towards substrates with a basic group at the P-1 position [Hilaire, P.M.S., Alves, L.C., Sanderson, S.J., Mottram, J.C., Juliano, M.A., Juliano, L., Coombs, G.H. & Meldal M. (2000) Chem. Biochem. 1, 115-122], but we have also observed high affinity for peptides with hydrophobic residues at this position. in order to have substrates containing both features, we synthesized one series of internally quenched fluorogenic peptides derived from the sequence ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine, and substituted the Arg at the P-1 position with the following non-natural basic amino acids: 4-aminomethyl-phenylalanine (Amf), 4-guanidine-phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). for comparison, the series derived from ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine was also assayed with cruzain (the major cysteine proteinase of Trypanosoma cruzi), human cathepsin L and papain. the peptides ortho-amino-benzoyl-FAmfSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K-m = 12 000 mm(-1).s(-1)) and ortho-amino-benzoyl-FIafSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K-m = 27 000 mm(-1).s(-1)) were the best substrates for CPB2.8 Delta CTE. in contrast, ortho-amino-benzoyl-FAmaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine and ortho-amino-benzoyl-FAcaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine were very resistant and inhibited this enzyme with K-i values of 23 nM and 30 nM, respectively. Cruzain hydrolyzed quite well the substrates in this series with Amf, Ppa and Aca, whereas the peptide with Ama was resistant and inhibited cruzain with a K-i of 40 nM. Human cathepsin L presented an activity on these peptides very similar to that of CPB2.8 Delta CTE and papain hydrolyzed all the peptides with high efficiency. in conclusion, we have demonstrated that CPB2.8 Delta CTE has more restricted specificity at the S-1 subsite and it seems possible to design efficient inhibitors with amino acids such as Ama or Aca at the P-1 position.
Palavra-chave cathepsin L
cruzain
cysteine proteinase of Leishmania mexicana
papain
Idioma Inglês
Data de publicação 2001-03-01
Publicado em European Journal of Biochemistry. Oxford: Blackwell Science Ltd, v. 268, n. 5, p. 1206-1212, 2001.
ISSN 0014-2956 (Sherpa/Romeo, fator de impacto)
Publicador Blackwell Science Ltd
Extensão 1206-1212
Fonte http://dx.doi.org/10.1046/j.1432-1327.2001.01973.x
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:000167601900006
Endereço permanente http://repositorio.unifesp.br/handle/11600/26494

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