Please use this identifier to cite or link to this item: http://hdl.handle.net/10316/3864
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dc.contributor.authorMiranda, Frederico F.-
dc.contributor.authorKolberg, Matthias-
dc.contributor.authorAndersson, K. Kristoffer-
dc.contributor.authorGeraldes, Carlos F. G. C.-
dc.contributor.authorMartínez, Aurora-
dc.date.accessioned2008-08-29T15:35:14Z-
dc.date.available2008-08-29T15:35:14Z-
dc.date.issued2005en_US
dc.identifier.citationJournal of Inorganic Biochemistry. 99:6 (2005) 1320-1328en_US
dc.identifier.urihttp://hdl.handle.net/10316/3864-
dc.description.abstractPhenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH4)-dependent enzyme that catalyzes the hydroxylation of l-Phe to l-Tyr. The non-heme iron in the enzyme (Fe(III) as isolated) is 6-coordinated to a 2-His-1-carboxylate motif and three water molecules (wat1, wat2 and wat3). Tyr325 is at the second coordination sphere, hydrogen-bonded to water (wat1). We prepared and expressed mutants with Leu, Ala, Ser and Phe at this position. Only Y325L and the conservative mutation Y325F resulted in stable enzymes, but the mutant Y325F has been found to be post-translationally hydroxylated and to revert back to wild-type PAH [S.D. Kinzie, M. Thevis, K. Ngo, J. Whitelegge, J.A. Loo, M.M. Abu-Omar, J. Am. Chem. Soc. 125 (2003) 4710-4711], being inadequate to investigate the early inferred functional role of Tyr325. On the other hand, compared to wild-type PAH, Y325L shows reduced specific activity, decreased coupling efficiency and decreased iron content. The mutant also reveals a very high affinity for l-Phe and BH4 and does not manifest positive cooperativity for the substrate. All together, our results support that the mutation Y325L causes the removal or increased delocalization of the iron-ligated wat1 and, in turn, a less tight binding of the metal. Tyr325 thus appears to have an important role ensuring stoichiometric binding of iron, correct geometry of the complexes with substrate and cofactor and, consequently, a right coupling efficiency of the PAH reaction. In addition, the residue appears to be important for the correct cooperative regulation by l-Phe.en_US
dc.description.urihttp://www.sciencedirect.com/science/article/B6TGG-4G24X8N-2/1/c2de94cf547830652188dae1c1f6ed94en_US
dc.format.mimetypeaplication/PDFen
dc.language.isoengeng
dc.rightsopenAccesseng
dc.subjectPhenylalanine hydroxylaseen_US
dc.subjectNon-heme ironen_US
dc.subjectCoupling efficiencyen_US
dc.subjectCoordination sphere, EPRen_US
dc.subjectTetrahydrobiopterinen_US
dc.titleThe active site residue tyrosine 325 influences iron binding and coupling efficiency in human phenylalanine hydroxylaseen_US
dc.typearticleen_US
dc.identifier.doi10.1016/j.jinorgbio.2005.03.005-
uc.controloAutoridadeSim-
item.fulltextCom Texto completo-
item.grantfulltextopen-
item.languageiso639-1en-
crisitem.author.deptFaculty of Sciences and Technology-
crisitem.author.parentdeptUniversity of Coimbra-
crisitem.author.researchunitCQC - Coimbra Chemistry Centre-
crisitem.author.parentresearchunitFaculty of Sciences and Technology-
crisitem.author.orcid0000-0002-0837-8329-
Appears in Collections:FCTUC Ciências da Vida - Artigos em Revistas Internacionais
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