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

Author 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
Institution Universidade Federal de São Paulo (UNIFESP)
Univ Glasgow
Univ Naples
Abstract 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.
Keywords cathepsin L
cruzain
cysteine proteinase of Leishmania mexicana
papain
Language English
Date 2001-03-01
Published in European Journal of Biochemistry. Oxford: Blackwell Science Ltd, v. 268, n. 5, p. 1206-1212, 2001.
ISSN 0014-2956 (Sherpa/Romeo, impact factor)
Publisher Blackwell Science Ltd
Extent 1206-1212
Origin http://dx.doi.org/10.1046/j.1432-1327.2001.01973.x
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000167601900006
URI http://repositorio.unifesp.br/handle/11600/26494

Show full item record




File

File Size Format View

There are no files associated with this item.

This item appears in the following Collection(s)

Search


Browse

Statistics

My Account