The role of Tyr(605) and Ala(607) of thimet oligopeptidase and Tyr(606) and Gly(608) of neurolysin in substrate hydrolysis and inhibitor binding

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dc.contributor.author Machado, Mauricio F. M. [UNIFESP]
dc.contributor.author Rioli, Vanessa
dc.contributor.author Dalio, Fernanda M.
dc.contributor.author Castro, Leandro M.
dc.contributor.author Juliano, Maria Aparecida [UNIFESP]
dc.contributor.author Tersariol, Ivarne L.
dc.contributor.author Ferro, Emer S.
dc.contributor.author Juliano, Luiz [UNIFESP]
dc.contributor.author Oliveira, Vitor [UNIFESP]
dc.date.accessioned 2016-01-24T13:48:44Z
dc.date.available 2016-01-24T13:48:44Z
dc.date.issued 2007-06-01
dc.identifier http://dx.doi.org/10.1042/BJ20070060
dc.identifier.citation Biochemical Journal. London: Portland Press Ltd, v. 404, p. 279-288, 2007.
dc.identifier.issn 0264-6021
dc.identifier.uri http://repositorio.unifesp.br/handle/11600/29762
dc.description.abstract The physicochemical properties of TOP (thimet oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series AbzGFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X = Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr(605) and Ala(607) in TOP and at Tyr(606) and Gly(608) in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. the kinetic parameters for the hydrolysis of substrates with systematic variations at position P-1 showed that Tyr(605) and Tyr(606) of TOP and NEL respectively, played a role in subsite S-1. Ala(607) of TOP and Gly(608) of NEL contributed to the flexibility of the loops formed by residues 600-612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599-611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P-1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Ty,105 was an important anchor for its interaction with wild-type TOP. the hydroxy groups of Tyr 605 and Tyr.. did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH-k(cat)/K-m dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis. en
dc.format.extent 279-288
dc.language.iso eng
dc.publisher Portland Press Ltd
dc.relation.ispartof Biochemical Journal
dc.rights Acesso aberto
dc.subject neurolysin en
dc.subject substrate and inhibitor specificity en
dc.subject site-directed mutagenesis en
dc.subject thimet oligopeptidase en
dc.title The role of Tyr(605) and Ala(607) of thimet oligopeptidase and Tyr(606) and Gly(608) of neurolysin in substrate hydrolysis and inhibitor binding en
dc.type Artigo
dc.contributor.institution Universidade Federal de São Paulo (UNIFESP)
dc.contributor.institution Inst Butantan
dc.contributor.institution Universidade de São Paulo (USP)
dc.contributor.institution Univ Mogi das Cruzes
dc.description.affiliation Universidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, Brazil
dc.description.affiliation Inst Butantan, Lab Especial Toxinol Aplicada, CAT, CEPID, BR-05467010 São Paulo, Brazil
dc.description.affiliation Univ São Paulo, Inst Ciencias Biomed, Dept Biol Celular & Desenvolvimento, Programa Biol Celular, BR-05508900 São Paulo, Brazil
dc.description.affiliation Univ São Paulo, Lab Neurociencias, BR-03071000 São Paulo, Brazil
dc.description.affiliation Univ Mogi das Cruzes, CIIB, BR-08780911 Mogi Das Cruzes, SP, Brazil
dc.description.affiliationUnifesp Universidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, Brazil
dc.identifier.doi 10.1042/BJ20070060
dc.description.source Web of Science
dc.identifier.wos WOS:000247014700012



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