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dc.contributor.authorLorenzon, Ricardo Z. [UNIFESP]
dc.contributor.authorCunha, Carlos E. L. [UNIFESP]
dc.contributor.authorMarcondes, Marcelo F. [UNIFESP]
dc.contributor.authorMachado, Mauricio F. M. [UNIFESP]
dc.contributor.authorJuliano, Maria A. [UNIFESP]
dc.contributor.authorOliveira, Vitor [UNIFESP]
dc.contributor.authorTravassos, Luiz R. [UNIFESP]
dc.contributor.authorPaschoalin, Thaysa [UNIFESP]
dc.contributor.authorCarmona, Adriana K. [UNIFESP]
dc.identifier.citationArchives of Biochemistry and Biophysics. New York: Elsevier B.V., v. 500, n. 2, p. 131-136, 2010.
dc.description.abstractOligopeptidase A (OpdA) belongs to the M3A subfamily of bacterial peptidases with catalytic and structural properties similar to mammalian thimet-oligopeptidase (TOP) and neurolysin (NEL). the three enzymes have four conserved Tyr residues on a flexible loop in close proximity to the catalytic site. in OpdA, the flexible loop is formed by residues 600-614 ((600)SHIFAGGYAAGYYSY(614)). Modeling studies indicated that in OpdA the Tyr(607) residue might be involved in the recognition of the substrate with a key role in catalysis. Two mutants were constructed replacing Tyr(607) by Phe (Y607F) or Ala (Y607A) and the influence of the site-directed mutagenesis in the catalytic process was examined. the hydrolysis of Abz-GXSPFRQ-EDDnp derivatives (Abz = ortho-aminobenzoic acid; EDDnp N-[2,4-dinitrophenyl]-ethylenediamine; X = different amino acids) was studied to compare the activities of wild-type OpdA (OpdA WT) and those of Y607F and Y607A mutants the results indicated that OpdA WT cleaved all the peptides only on the X-S bond whereas the Y607F and Y607A mutants were able to hydrolyze both the X-S and the P-F bonds. the kinetic parameters showed the importance of Tyr(607) in OpdA catalytic activity as its substitution promoted a decrease in the k(cat)/K-m value of about 100-fold with Y607F mutant and 1000-fold with Y607A. Both mutations, however, did not affect protein folding as indicated by CD and intrinsic fluorescence analysis. Our results indicate that the OpdA Tyr(607) residue plays an important role in the enzyme-substrate interaction and in the hydrolytic activity. (C) 2010 Elsevier Inc. All rights reserved.en
dc.description.sponsorshipConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.publisherElsevier B.V.
dc.relation.ispartofArchives of Biochemistry and Biophysics
dc.rightsAcesso restrito
dc.subjectM3A subfamilyen
dc.subjectE. coli oligopeptididase Aen
dc.subjectSite-directed mutagenesisen
dc.subjectFRET substratesen
dc.titleKinetic characterization of the Escherichia coli oligopeptidase A (OpdA) and the role of the Tyr(607) residueen
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.description.affiliationUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, Brazil
dc.description.affiliationUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, Brazil
dc.description.sourceWeb of Science
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