Activation of Phenylalanine Hydroxylase by Phenylalanine Does Not Require Binding in the Active Site

Roberts, Kenneth M.; Khan, Crystal A.; Hinck, Cynthia S.; Fitzpatrick, Paul F.

doi:10.1021/bi501183x

Cited by 23 publications

(36 citation statements)

References 48 publications

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“…2B, colored circles in inset). The mid-point of the transition for the PheH R270K mutant is shifted relative to the wild-type enzyme, occurring at a [L-Phe] of 370 ± 35 μ M. A similar shift was previously observed by tryptophan fluorescence measurements of the same construct 26 . As a superposition of our PheH Δ24 R270K and wt-PheH structures shows no significant change beyond the immediate vicinity of the mutation (Fig.…”

Section: Resultssupporting

confidence: 84%

“…First, the allosteric binding site for L-Phe was shown by mutagenesis and fluorescence quenching to be distinct from the active site. 26 In this study, an R270K mutation, which has been associated with PKU in humans, 27 was shown to abolish L-Phe binding in the active site in the full-length enzyme ( K m > 0.5 M), while retaining the ability to be activated by L-Phe. Furthermore, recent nuclear magnetic resonance (NMR) and analytical ultracentrifugation studies establish that a PheH construct containing only the regulatory domain undergoes a monomer-dimer transition in the presence of L-Phe 28,29 and that two molecules of L-Phe bind at a canonical ACT-domain dimer interface.…”

Section: Introductionmentioning

confidence: 62%

“…S4A). The effect of the R270K mutation is localized to the loop containing Thr278; substitution of Arg270 with Lys270 not only eliminates ionic interactions that support binding of L-Phe in the active site, 26 but also introduces a new hydrogen bond with the backbone, which in turn appears to favor an alternate conformation of the Thr278 loop (Fig. S4B–C).…”

Section: Resultsmentioning

confidence: 99%

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Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X-ray Scattering

et al. 2016

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Mammalian phenylalanine hydroxylase (PheH) is an allosteric enzyme that catalyzes the first step in the catabolism of the amino acid phenylalanine. Following allosteric activation by high phenylalanine levels, the enzyme catalyzes the pterin-dependent conversion of phenylalanine to tyrosine. Inability to control elevated phenylalanine levels in the blood leads to increased risk of mental disabilities commonly associated with the inherited metabolic disorder, phenylketonuria. Although extensively studied, structural changes associated with allosteric activation in mammalian PheH have been elusive. Here, we examine the complex allosteric mechanisms of rat PheH using X-ray crystallography, isothermal titration calorimetry (ITC), and small-angle X-ray scattering (SAXS). We describe crystal structures of the pre-activated state of the PheH tetramer depicting the regulatory domains docked against the catalytic domains and preventing substrate binding. Using SAXS, we further describe the domain movements involved in allosteric activation of PheH in solution and present the first demonstration of chromatography-coupled SAXS with Evolving Factor Analysis (EFA), a powerful method for separating scattering components in a model-independent way. Together, these results support a model for allostery in PheH in which phenylalanine stabilizes the dimerization of the regulatory domains and exposes the active site for substrate binding and other structural changes needed for activity.

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Section: Resultssupporting

confidence: 84%

Section: Introductionmentioning

confidence: 62%

Section: Resultsmentioning

confidence: 99%

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Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X-ray Scattering

et al. 2016

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“…Because we introduced the idea that an activated tetramer contains an ACT domain dimer and that this dimer interface is the site of allosteric Phe binding (12), several new studies have provided support for this concept (27)(28)(29). We suggest a model for the activated PAH tetramer (Fig.…”

Section: Discussionmentioning

confidence: 97%

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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“…Recent studies of the isolated regulatory domain of rat PheH (RDPheH) have established that this domain indeed binds phenylalanine (23)(24)(25). Moreover, elimination of phenylalanine binding in the active site does not prevent the conformational change associated with phenylalanine activation, consistent with an allosteric site separate from the active site (26).…”

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confidence: 99%

Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase

Zhang

Fitzpatrick

2016

Journal of Biological Chemistry

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Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25-117) is the regulatory domain of PheH lacking residues 1-24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25-117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25-117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH. Phenylalanine hydroxylase (PheH)2 catalyzes a key step in phenylalanine catabolism, the hydroxylation of phenylalanine to tyrosine in the liver using tetrahydropterin (BH 4 ) and oxygen. A deficiency in human PheH increases the level of phenylalanine in the blood, resulting in the inherited disease phenylketonuria (PKU) (1). Thus, the activity of PheH must be tightly controlled to maintain appropriate phenylalanine levels. PheH is activated by phenylalanine and inhibited by BH 4 (2, 3). In addition, phosphorylation of PheH at Ser-16 is reported to decrease the concentration of phenylalanine required to activate PheH (4).Mammalian PheH is a homotetramer, and each monomer contains an N-terminal regulatory domain, a central catalytic domain, and a C-terminal tetramerization domain. The other two aromatic amino acid hydroxylases, tyrosine hydroxylase (TyrH) and tryptophan hydroxylase, have similar architectures.The crystal structures of the catalytic domains of all three enzymes show very similar folds and active sites (5-7), consistent with these enzymes sharing a common catalytic mechanism (8). The structures of the regulatory domains of PheH and TyrH show that both contain ACT domains (5, 9, 10), although the two enzymes are regulated differently (2, 11). To date, there is no published structure of a full-length mammalian PheH, or indeed of any eukaryotic aromatic amino acid hydroxylase, in that the available structures are of proteins lacking the N-terminal regulatory domain, much of the C-terminal tetramerization domain, or both. The structure of a dimeric form of rat PheH containing both the catalytic and regulatory domains but lacking the C-terminal 24 residues required for tetramer formation (5) has provided the present model for the structural basis for activation by phenylalanine. In this structure the...

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Activation of Phenylalanine Hydroxylase by Phenylalanine Does Not Require Binding in the Active Site

Cited by 23 publications

References 48 publications

Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X-ray Scattering

Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X-ray Scattering

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

Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase

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