The Phenylalanine Hydroxylating System

Kaufman, Seymour

doi:10.1002/9780470123133.ch2

Cited by 80 publications

(95 citation statements)

References 271 publications

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“…Phosphorylation at Ser16 potentiates the effects of Phe, with phosphorylated PAH achieving full activation at lower Phe concentrations than the unphosphorylated protein (5,6). Allosteric activation by Phe is accompanied by a major conformational change, as evidenced by changes in protein fluorescence and proteolytic susceptibility, and by stabilization of a tetrameric conformer (3).…”

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confidence: 99%

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First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

Arturo

Gupta

Héroux

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

122

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Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73-82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype-phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phestabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype-phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate.phenylalanine hydroxylase | phenylketonuria | X-ray crystallography | small-angle X-ray scattering | allosteric regulation M ammalian phenylalanine hydroxylase (PAH) (EC 1.14.16.1) is a multidomain homo-multimeric protein whose dysfunction causes the most common inborn error in amino acid metabolism, phenylketonuria (PKU), and milder forms of hyperphenylalaninemia (OMIM 261600) (1). PAH catalyzes the hydroxylation of phenylalanine (Phe) to tyrosine, using nonheme iron and the cosubstrates tetrahydrobiopterin and molecular oxygen (2, 3). A detailed kinetic mechanism has recently been derived from elegant single-turnover studies (4). PAH activity must be carefully regulated, because although Phe is an essential amino acid, high Phe levels are neurotoxic. Thus, Phe allosterically activates PAH by binding to a regulatory domain. Phosphorylation at Ser16 potentiates the effects of Phe, with phosphorylated PAH achieving full activation at lower Phe concentrations than the unphosphorylated protein (5, 6). Allosteric activation by Phe is accompanied by a major conformational change, as evidenced by changes in protein fluorescence and proteolytic susceptibility, and by stabilization of a tetrameric confo...

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mentioning

confidence: 99%

“…PAH catalyzes the hydroxylation of phenylalanine (Phe) to tyrosine, using nonheme iron and the cosubstrates tetrahydrobiopterin and molecular oxygen (2,3). A detailed kinetic mechanism has recently been derived from elegant single-turnover studies (4).…”

mentioning

confidence: 99%

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

Arturo

Gupta

Héroux

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

122

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“…The slight differences found were due to the fact that previously reported PAH activity measurements were performed under different assay conditions and using several substrates with different affinities (e.g. BH 4 , 6-methyl-tetrahydropterin or 6,7-dimethyltetrahydropterin) [3,12]. In previous reports, in vitro activities for some mutations (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…The phenotypic severity of PKU is characterized by the type of mutation, and thus by residual PAH enzyme activity. The fully functional homotetrameric PAH catalyzes hydroxylation of Phe to tyrosine (Tyr) in the presence of cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH 4 ) and molecular oxygen [3,4]. According to the Locus Knowledgebase (PAHdb, www.pahdb.mcgill.ca), about 60% of mutations in the PAH gene are missense mutations, which may lead to a misfolding of the protein [5,6], disturbing the complex enzyme regulation and changes in kinetics, due to altered affinities for the Phe substrate and the BH 4 cofactor.…”

Section: Introductionmentioning

confidence: 99%

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Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography–electrospray ionization tandem mass spectrometry

Heintz

Troxler

Martı́nez

et al. 2012

Molecular Genetics and Metabolism

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BACKGROUND: Residual phenylalanine hydroxylase (PAH) activity is the key determinant for the phenotype severity in phenylketonuria (PKU) patients and correlates with the patient's genotype. Activity of in vitro expressed mutant PAH may predict the patient's phenotype and response to tetrahydrobiopterin (BH(4)), the cofactor of PAH. METHODS: A robust LC-ESI-MSMS PAH assay for the quantification of phenylalanine and tyrosine was developed. We measured PAH activity a) of the PAH mutations p.Y417C, p.I65T, p.R261Q, p.E280A, p.R158Q, p.R408W, and p.E390G expressed in eukaryotic COS-1 cells; b) in different cell lines (e.g. Huh-7, Hep3B); and c) in liver, brain, and kidney tissue from wild-type and PKU mice. RESULTS: The PAH assay was linear for phenylalanine and tyrosine (r(2) 0.99), with a detection limit of 105 nmol/L for Phe and 398 nmol/L for Tyr. Intra-assay and inter-assay coefficients of variation were <5.3% and <6.2%, respectively, for the p.R158Q variant in lower tyrosine range. Recovery of tyrosine was 100%. Compared to the wild-type enzyme, the highest PAH activity at standard conditions (1 mmol/L L-Phe; 200 mol/L BH(4)) was found for the mutant p.Y417C (76%), followed by p.E390G (54%), p.R261Q (43%), p.I65T (33%), p.E280A (15%), p.R158Q (5%), and p.R408W (2%). A relative high PAH activity was found in kidney (33% of the liver activity), but none in brain. CONCLUSIONS: This novel method is highly sensitive, specific, reproducible, and efficient, allowing the quantification of PAH activity in different cells or tissue extracts using minimum amounts of samples under standardized conditions

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Pterin Carbinolamine Dehydratase/Dimerization Cofactor ofHNF1

Kaufman

Lei

2002

Wiley Encyclopedia of Molecular Medicine

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The Phenylalanine Hydroxylating System

Cited by 80 publications

References 271 publications

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography–electrospray ionization tandem mass spectrometry

Pterin Carbinolamine Dehydratase/Dimerization Cofactor ofHNF1

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