The substrate specificity of a recombinant cysteine protease from Leishmania mexicana: Application of a combinatorial peptide library approach

The substrate specificity of a recombinant cysteine protease from Leishmania mexicana: Application of a combinatorial peptide library approach

Autor St Hilaire, P. M. Google Scholar
Alves, L. C. Google Scholar
Sanderson, S. J. Google Scholar
Mottram, J. C. Google Scholar
Juliano, M. A. Google Scholar
Juliano, L. Google Scholar
Coombs, G. H. Google Scholar
Meldal, M. Google Scholar
Instituição Carlsberg Lab
Universidade Federal de São Paulo (UNIFESP)
Univ Glasgow
Resumo The substrate specificity of CPB2.8 Delta CTE, a recombinant cysteine protease from Leishmania mexicana, was mapped by screening a fluorescence-quenched combinatorial peptide library. Results from library screening indicated a preference for Arg or Lys in the S-3 subsite and for hydrophobic residues, both aliphatic and aromatic, in S-2. the S-1 subsite exhibited a specificity for the basic residues Arg and Lys. Generally, the specificity of the primed subsites was less strict compared with the non-primed side which showed preference for Arg, Lys and Ala in S-1, Arg, Pro and Gly in S-2 and Lys, Arg and Ser in S-4. By contrast, a strict preference for the basic residues Arg and Lys was found for S-3. Overall, there was a trend for basic residues in alternating subsites and smaller residues in the primed sites compared with the non-primed sites. in addition, there were strict requirements for the amino acids in subsites S-3-S-1. Fluorescence-quenched peptides from the library with the highest on-resin cleavage were resynthesised and the their kinetics of hydrolysis by CPB1.8 Delta CTE assessed in solution phase assays. Several good substances containing the quintessential dipeptide particular to cathespin-L-like enzymes, -F-R/K-, in P-2 and P-1 were identified (e.g. Y(NO2)-EKFR down arrow RGK-K(Abz)G, Abz = 2-aminobenzoyl; k(car)K(m) (1) = 4298 mM(-1) s(1)). However, novel substrates containing the dipeptide -L/I-Q- in P-2 and P-1 were also well hydrolysed (e.g. Y(NO2)-YLQ down arrow GIQK-K(Abz)G; k(cat)K(m)(-1) = 2583 mM(-1) s-(1)). the effect of utilising different fluorescent donor-quencher pairs on the value of k(cat)K(m)(-1) was examined. Generally, the use of the Abz/Q-EDDnp donor-quencher pair (EDDnp = N-(2,4-dinitrophenyl)ethylenediamine) instead of K(Abz)/Y(NO2) resulted in higher k(cat)K(m)(-1) values for analogous substrates.
Palavra-chave combinatorial chemistry
enzyme catalysis
fluorescence resonance energy transfer
Idioma Inglês
Data de publicação 2000-08-18
Publicado em Chembiochem. Weinheim: Wiley-v C H Verlag Gmbh, v. 1, n. 2, p. 115-122, 2000.
ISSN 1439-4227 (Sherpa/Romeo, fator de impacto)
Publicador Wiley-Blackwell
Extensão 115-122
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000165767300004
Endereço permanente

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