Defining the substrate specificity of mouse cathepsin P

Defining the substrate specificity of mouse cathepsin P

Autor Puzer, L. Google Scholar
Barros, NMT Google Scholar
Oliveira, V Google Scholar
Juliano, M. A. Google Scholar
Lu, G. Z. Google Scholar
Hassanein, M. Google Scholar
Juliano, L. Google Scholar
Mason, R. W. Google Scholar
Carmona, A. K. Google Scholar
Instituição Universidade Federal de São Paulo (UNIFESP)
Univ Cidade São Paulo
Alfred I DuPont Hosp Children
Univ Delaware
Resumo Cathepsin P is a recently discovered placental cysteine protease that is structurally related to the more ubiquitously expressed, broad-specificity enzyme, cathepsin L. We studied the substrate specificity requirements of recombinant mouse cathepsin P using fluorescence resonance energy transfer (FRET) peptides derived from the lead sequence Abz-KLRSSKQ-EDDnp (Abz, ortho-aminobenzoic acid and EDDnp, N-[2,4-dinitroplieiiyl]ethyleiiediamine). Systematic modifications were introduced resulting in five series of peptides to map the S-3 to S-2' subsites of the enzyme. the results indicate that the subsites S-1, S-2, S-1' and S-2', present a clear preference for hydrophobic residues. the specificity requirements of the S, subsite were found to be more restricted, preferring hydrophobic aliphatic amino acids. the S-3 subsite of the enzyme presents a broad specificity, accepting negatively charged (Glu), positively charged (Lys, Arg), and hydrophobic aliphatic or aromatic residues (Val, Phe). for several substrates, the activity of cathepsin P was markedly regulated by kosmotropic salts, particularly Na2SO4. No significant effect on secondary or tertiary structure could be detected by either circular dichroism or size exclusion chromatography, indicating that the salts most probably disrupt unfavorable ionic interactions between the substrate and enzyme active site. A substrate based upon the preferred P-3 to P-2' defined by the screening study, ortho-aminobenzoic-Glu-Ile-Phe-Val-Phe-Lys-Gln-N-(2,4-dinitrophenyl)ethylenediamine (cleaved at the Phe-Val bond) was efficiently hydrolyzed in the absence of high salt. the k(cat)/K-m for this substrate was almost two orders of magnitude higher than that of the original parent compound. These results show that cathepsin P, in contrast to other mammalian cathepsins, has a restricted catalytic specificity. (C) 2004 Elsevier Inc. All rights reserved.
Palavra-chave cysteine proteases
placentally expressed cathepsins
cathepsin P
flurogenic substrates
substrate specificity
Idioma Inglês
Data de publicação 2005-03-01
Publicado em Archives of Biochemistry and Biophysics. New York: Elsevier B.V., v. 435, n. 1, p. 190-196, 2005.
ISSN 0003-9861 (Sherpa/Romeo, fator de impacto)
Publicador Elsevier B.V.
Extensão 190-196
Fonte http://dx.doi.org/10.1016/j.abb.2004.12.007
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000226782400021
Endereço permanente http://repositorio.unifesp.br/handle/11600/28174

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