Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity

Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity

Autor St Hilaire, P. M. Google Scholar
Willert, M. Google Scholar
Juliano, M. A. Google Scholar
Juliano, L. Google Scholar
Meldal, M. Google Scholar
Instituição Dept Chem
Universidade Federal de São Paulo (UNIFESP)
Resumo To map the substrate specificity of cysteine proteases, two combinatorial peptide libraries were synthesized and screened using the archetypal protease, papain. the use of PEGA resin as the solid support for library synthesis facilitated the application of an on-resin fluorescence-quenched assay. Results from the screening of library 2 indicated a preference for Pro or Val in the S-3 subsite and hydrophobic residues in S-2; the most prevalent residue not being Phe but Val. the S-1 subsite exhibited a dual specificity for both small, nonpolar residues, Ala or Gly, as well as larger, Gin, and charged residues, Arg. Small residues predominated in the S-1'-S-4' subsites. Active peptides from the libraries and variations thereof were resynthesized and their kinetics of hydrolysis by papain assessed in solution phase assays, Generally, there was a good correlation between the extent of substrate cleavage on solid phase and the k(cat)/K-M's obtained in solution phase assays. Several good substrates for papain were obtained, the best substrates being Y(NO2)PMPPLCTSMK(Abz) (k(cat)/K-M = 2109 (mM s)(-1)), Y(NO2)PYAVQSPQK(Abz) (k(cat)/K-M = 1524 (mM s)(-1)), and Y(NO2)PVLRQQRSK(Abz) (k(cat)/K-M = 1450 (mM s)(-1)). These results were interpreted in structural terms by the use of molecular dynamics (MD). These MD calculations indicated two different modes for the binding of substrates in the narrow enzyme cleft.
Idioma Inglês
Data de publicação 1999-11-01
Publicado em Journal of Combinatorial Chemistry. Washington: Amer Chemical Soc, v. 1, n. 6, p. 509-523, 1999.
ISSN 1520-4766 (Sherpa/Romeo, fator de impacto)
Publicador Amer Chemical Soc
Extensão 509-523
Fonte http://dx.doi.org/10.1021/cc990031u
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000084203900009
Endereço permanente http://repositorio.unifesp.br/handle/11600/26174

Exibir registro completo




Arquivo

Arquivo Tamanho Formato Visualização

Não existem arquivos associados a este item.

Este item está nas seguintes coleções

Buscar


Navegar

Minha conta