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

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dc.contributor.authorSong, E. S.
dc.contributor.authorMukherjee, A.
dc.contributor.authorJuliano, Maria Aparecida [UNIFESP]
dc.contributor.authorSt Pyrek, J.
dc.contributor.authorGoodman, J. P.
dc.contributor.authorJuliano, Luiz [UNIFESP]
dc.contributor.authorHersh, L. B.
dc.contributor.institutionUniv Kentucky
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.date.accessioned2016-01-24T12:31:17Z
dc.date.available2016-01-24T12:31:17Z
dc.date.issued2001-01-12
dc.description.abstractRecombinant 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.en
dc.description.affiliationUniv Kentucky, Albert B Chandler Med Ctr, Coll Med, Dept Biochem, Lexington, KY 40536 USA
dc.description.affiliationUniv Kentucky, Mass Spectrometry Facil, Lexington, KY 40506 USA
dc.description.affiliationEscola Paulista Med, Dept Biophys, BR-04034 São Paulo, Brazil
dc.description.affiliationUnifespEscola Paulista Med, Dept Biophys, BR-04034 São Paulo, Brazil
dc.description.sourceWeb of Science
dc.format.extent1152-1155
dc.identifierhttp://dx.doi.org/10.1074/jbc.M008702200
dc.identifier.citationJournal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 276, n. 2, p. 1152-1155, 2001.
dc.identifier.doi10.1074/jbc.M008702200
dc.identifier.issn0021-9258
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/26471
dc.identifier.wosWOS:000166430900042
dc.language.isoeng
dc.publisherAmer Soc Biochemistry Molecular Biology Inc
dc.relation.ispartofJournal of Biological Chemistry
dc.rightsinfo:eu-repo/semantics/openAccess
dc.titleAnalysis of the subsite specificity of rat insulysin using fluorogenic peptide substratesen
dc.typeinfo:eu-repo/semantics/article
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