Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P-4, P-3, P-2 ' and P-3 ' variations in extended substrates

Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P-4, P-3, P-2 ' and P-3 ' variations in extended substrates

Autor Portaro, Fernanda Calheta Vieira Autor UNIFESP Google Scholar
Santos, Ana Beatriz F Google Scholar
Cezari, Maria Helena S Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Carmona, Euridice Google Scholar
Instituição Inst Butantan
Universidade Federal de São Paulo (UNIFESP)
Resumo We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S-4 and S-3 subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S-2' and S-3'. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P-4, P-3, P-2' and P-3' were made. the S-4 to S-2' subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S-3', indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S-4, S-3, S-3' and S-3' of the three enzymes. the best substrates for cathepsins L and B had Trp and Asn at P-2' respectively; variations at this position were less accepted by these enzymes. the best substrates for papain were peptides containing Trp, Tyr or Asn at P-3'. Basic residues at P-3 and p(4) were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P-2' (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. the modifications at His(111) (H111A) and His(110) (H110A) of cathepsin B led to an increase in k(cat) values of one or two orders of magnitude. the hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.
Palavra-chave cathepsins B and L
fluorogenic peptide
limited proteolysis
papain
protease substrate
Idioma Inglês
Data de publicação 2000-04-01
Publicado em Biochemical Journal. London: Portland Press, v. 347, p. 123-129, 2000.
ISSN 0264-6021 (Sherpa/Romeo, fator de impacto)
Publicador Portland Press
Extensão 123-129
Fonte http://dx.doi.org/10.1042/0264-6021:3470123
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:000086792600016
Endereço permanente http://repositorio.unifesp.br/handle/11600/26274

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