Analysis of the subsite specificity of rat insulysin using fluorogenic peptide substrates

Analysis of the subsite specificity of rat insulysin using fluorogenic peptide substrates

Autor Song, E. S. Google Scholar
Mukherjee, A. Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
St Pyrek, J. Google Scholar
Goodman, J. P. Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Hersh, L. B. Google Scholar
Instituição Univ Kentucky
Universidade Federal de São Paulo (UNIFESP)
Resumo Recombinant rat insulysin was shown to cleave the internally quenched fluorogenic peptide 2-aminobenzyl-GGFLRKVGQ-ethylenediamine-2,4-dinitrophenol at the R-K bond, exhibiting a K-m of 13 muM and a V-max of 2.6 mu mol min(-1) mg(-1). Derivatives of this peptide in which the P-2 leucine or the P-2' valine were replaced with other residues were used to probe the subsite specificity of the enzyme. Varying the P-2 residue produced a l-fold range in K-m and a 7-fold range in k(cat). the nature of the P-2 residue had a significant effect on the site of cleavage. Leucine, isoleucine, valine, and aspartate produced cleavage at the R-K bond, Asparagine produced 36% cleavage at the N-R bond and 64% cleavage at the RK bond, whereas with alanine or serine the A-R and S-R bonds were the major cleavage sites. With tyrosine, phenylalanine, methionine, or histidine representing the varied residue X, cleavages at F-X, X-R, and RK were seen, whereas with tryptophan equal cleavage occurred at the F-W and W-R bonds. Variable P-2' residues produce less of a change in both K-m and k(cat) and have little influence on the cleavage site. Exceptions are phenylalanine, tyrosine, leucine, and isoleucine, which in addition to producing cleavage at the RK bond, produce significant cleavage at the GR bond. Alanine and tyrosine were unique in producing cleavage at the F-L bond. Taken together, these data suggest that insulysin specificity is directed toward the amino side of hydrophobic and basic residues and that the enzyme has an extended substrate binding site.
Idioma Inglês
Data de publicação 2001-01-12
Publicado em Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 276, n. 2, p. 1152-1155, 2001.
ISSN 0021-9258 (Sherpa/Romeo, fator de impacto)
Publicador Amer Soc Biochemistry Molecular Biology Inc
Extensão 1152-1155
Fonte http://dx.doi.org/10.1074/jbc.M008702200
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:000166430900042
Endereço permanente http://repositorio.unifesp.br/handle/11600/26471

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