Mutation of active site residues of insulin-degrading enzyme alters allosteric interactions

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dc.contributor.author Song, E. S.
dc.contributor.author Daily, A.
dc.contributor.author Fried, M. G.
dc.contributor.author Juliano, Maria Aparecida [UNIFESP]
dc.contributor.author Juliano, Luiz [UNIFESP]
dc.contributor.author Hersh, L. B.
dc.date.accessioned 2016-01-24T12:37:51Z
dc.date.available 2016-01-24T12:37:51Z
dc.date.issued 2005-05-06
dc.identifier http://dx.doi.org/10.1074/jbc.M501896200
dc.identifier.citation Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 280, n. 18, p. 17701-17706, 2005.
dc.identifier.issn 0021-9258
dc.identifier.uri http://repositorio.unifesp.br/handle/11600/28299
dc.description.abstract The active site glutamate (Glu(111)) and the active site histidine (His(112)) of insulin-degrading enzyme (IDE) were mutated. These mutant enzymes exhibit, in addition to a large decrease in catalytic activity, a change in the substrate-velocity response from a sigmoidal one seen with the native enzyme (Hill coefficient >2), to a hyperbolic response. With 2-aminobenzoyl-GGFLRKHGQN-(2,4-dinitrophenyl)ethylenediamine as substrate, ATP and triphosphate increase the reaction rate of the wild type enzyme some 50-80-fold. This effect is dampened with glutamate mutants to no effect or less than a 3-fold increase in activity and changed to inhibition with the histidine mutants. Sedimentation equilibrium shows the IDE mutants exhibit a similar oligomeric distribution as the wild type enzyme, being predominantly monomeric, with triphosphate having little if any effect on the oligomeric state. Triphosphate did induce aggregation of many of the IDE mutants. Thus, the oligomeric state of IDE does not correlate with kinetic properties. the His(112) mutants were shown to bind zinc, but with a lower affinity than the wild type enzyme. the glutamate mutants displayed an altered cleavage profile for the peptide beta-endorphin. Wild type IDE cleaved beta-endorphin at Leu(17)-Phe(18) and Phe(18)-Lys(19), whereas the glutamate mutants cleaved at these sites, but in addition at Lys(19)-Asn(20) and at Met(5)-Thr(6). Thus, active site mutations of IDE are suggested to not only reduce catalytic activity but also cause local conformational changes that affect the allosteric properties of the enzyme. en
dc.format.extent 17701-17706
dc.language.iso eng
dc.publisher Amer Soc Biochemistry Molecular Biology Inc
dc.relation.ispartof Journal of Biological Chemistry
dc.rights Acesso aberto
dc.title Mutation of active site residues of insulin-degrading enzyme alters allosteric interactions en
dc.type Artigo
dc.contributor.institution Univ Kentucky
dc.contributor.institution Universidade Federal de São Paulo (UNIFESP)
dc.description.affiliation Univ Kentucky, Dept Mol & Cellular Biochem, Coll Med, Lexington, KY 40536 USA
dc.description.affiliation Escola Paulista Med, Dept Biophys, BR-04023900 São Paulo, Brazil
dc.description.affiliationUnifesp Escola Paulista Med, Dept Biophys, BR-04023900 São Paulo, Brazil
dc.identifier.doi 10.1074/jbc.M501896200
dc.description.source Web of Science
dc.identifier.wos WOS:000228807200019



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