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The active site residue tyrosine 325 influences iron binding and coupling efficiency in human phenylalanine hydroxylase
- Source :
- Repositório Científico de Acesso Aberto de Portugal, Repositório Científico de Acesso Aberto de Portugal (RCAAP), instacron:RCAAP
- Publication Year :
- 2005
- Publisher :
- Elsevier BV, 2005.
-
Abstract
- Phenylalanine 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. http://www.sciencedirect.com/science/article/B6TGG-4G24X8N-2/1/c2de94cf547830652188dae1c1f6ed94
- Subjects :
- Models, Molecular
Phenylalanine hydroxylase
Stereochemistry
Iron
Mutant
DNA, Recombinant
Coordination sphere, EPR
In Vitro Techniques
Hydroxylation
Biochemistry
Cofactor
Inorganic Chemistry
chemistry.chemical_compound
Catalytic Domain
medicine
Humans
Tyrosine
Tetrahydrobiopterin
Base Sequence
biology
Electron Spin Resonance Spectroscopy
Phenylalanine Hydroxylase
Cooperative binding
Active site
Recombinant Proteins
Kinetics
Spectrometry, Fluorescence
chemistry
Mutagenesis, Site-Directed
biology.protein
Non-heme iron
Coupling efficiency
medicine.drug
Subjects
Details
- ISSN :
- 01620134
- Volume :
- 99
- Database :
- OpenAIRE
- Journal :
- Journal of Inorganic Biochemistry
- Accession number :
- edsair.doi.dedup.....afd20448c421eba09605876ce86bcda1
- Full Text :
- https://doi.org/10.1016/j.jinorgbio.2005.03.005