Review On Phenylalanine Hydroxylase Pheoh Biology Essay

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INTRODUCTION

Phenylalanine Hydroxylase (PheOH), alternatively PheH or PAH catalyzes the hydroxylation of the amino acid, phenylalanine to tyrosine. PheOH is a class of monooxygenase that uses a non-heme iron and tetrahydrobiopterin (BH4) for catalysis. PheOH functions as a protein regulator where 75% of phenylalanine in a person's liver is converted to ingested, while the phenylalanine left in the body unconverted may cause mental retardation. It is through the rapid degradation of Phe that can lead to neurological damage and decrease protein synthesis. Our control of enzyme within the levels of dietary intake of amino acids is shown in our bloodstream.

Lphenylalanine + BH4 + O2 PheH Ltyrosine + 4ahydroxyBH4

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Figure 1. Conversion of L-phenylalanine (Phe) to L-tyrosine (Tyr)

In figure 1, PheH converts Phe into tyrosine (Tyr) by hydroxylating Phe, using BH4 as a reductant. The reaction showed the adding of -OH to the group of the 6-ring carbon. The PheH is the monooxygenase where in the amino acid substrate, a molecule of oxygen incorporates, while using BH4 in the other oxygen, as a reductant, the atom is reduced.

The Structure: Phenylalanine Hydroxylase PheOH

The four subunits of PheOH is composed of 3 domains namely: a 12-19 kDa N-terminal regulation domain, a 33-kDa catalytic domain, and a 5-kDa C-terminal tetramerization domain. The tetramerization domain forms a ribbon and a long helix. A form of coiled-coil motif comes in contact with the each subunit of the helical arm. A 2-7 alpha helices forms a tightly intertwined protein. The subunits are pulled closer that formed the protein into a final quarternary structure, having the four coiled tetramerization domain at the center.

Figure 2.  Four subunits of PheOH

Phenylalanine Hydroxylase is regulated by phenylalanine, tetrahydrobiopterin, and phosphorylation according to Kobe, et al (1999). Figure 1 shows the single subunit of PheOH. The catalytic site is located at the center where the molecule of Phenylalanine is located. The C- Terminus of the monomer is responsible for tetramerization. Within the C-terminus helical walls, lies the active site. Meanwhile, the regulatory domain extending across the active site of the catalytic domain is the N-terminus, which is similar to phosphoglycerate dehydrogenase, in serine biosynthesis, and PCD, in BH4 regeneration and NHF1 activation which activates PAH transcription.

Figure 3. Structure of Phenylalanine Hydroxylase

The Three Distinct Domains of PheOH:

N-terminal regulatory domain, Catalytic domain, and C-Terminal Tetramerization domain

N-Terminal Regulatory Domain

Hydrogen exchanges states allosteric binding of Phe alters PheOH. Active site interface, regulatory and catalytic domains are exposed to solvent. This shows that Full length PheOH have a low rate of tyrosine. Elimination of lag time in deletion of N-terminal causes the affinity if Phe to increase by nearly two-fold.

For the tetrahydrobipterin cofactor in Vmax and Km, no difference is observed. Phosphorylation does not alter enzyme conformation while the concentration of Phe is reduce for allosteric activation. Structural homology to the domain of phosphoglycerate was shown.

Catalytic Domain

The active site consists an open pocket lined by hydrophobic residues. The three glutamic acid residues, two histidines, and tyrosine are critical for pterin- and iron-binding. The coordination state of the ferrous atom and BH4 is located at the active site.

Inlcusion of Phe in the crystal structure changes iron to a 5- coordinated state involving a singe H2O molecule and a bidentate coordination to Glu330. BH4 shifted toward the iron atom and Pterin cofactor remains. While, all coordinated water molecules are forced out the active site and BH4 is coordinated to iron.

C-Terminal Tetramerization Domain

Eukaryotic PheOH exists between homodimeric and homotetrameric forms. A symmetry related loops is a composition if a dimerization interface that links identical monomers. Tetramerization domain mediated the association of dimers that are relative to a different orientation of catalytic domains. The differential surface of dimerization results to a distortion of the tetramer symmetry and compares PheOH from tyrosine hydroxylase.

A domain swapping mechanism forms tetramer from dimers, where C-terminal alpha helixes alters around a flexible C-terminal 5-residue hinge region in forming a coiled-coil structure. Homodimeric and homotetrametric forms are differential in regulation and kinetics. In addition, L-Phe regulates PheOH having a dimer-dimer interaction.

Functions and Mechanisms of PheOH

Phenylalanine have two binding sites, a catalytic site and a regulatory site. The binding site causes the drastic shape change which results to a cleavage formation of the Phe substrate. In order for PheOH to catalyze Phe in dephosphorylated state, a high concentration is needed. N-terminus phosphorylation site may be altered when phosphorylated. Structural changes may be observed, the N-terminus is moved from the active site for Phe to bind with PheOH at a lower concentration.

Other point of PheOH regulation is, BH4 may bind to PheOH near a hinge site which gives of two hyphothesis to why BH4 inhibits PheOH. First, the structural change of PheOH hinge site may be hindered by BH4 when PheOH is bound with Phe. Second, N-terminus phosphorylation site movement may be block by BH4 which can result in a less active Phe binding site.

Another aspect is the mutations in PAH and their structural changes in enzyme. Many different mutations in PAH reduced the function of PheOH and PKU. Structural implications on active site are mutations of PheOH. However, the mutations in the N-terminus region and catalytic domain were identified. N-terminus function and hinge site regulation explains the decreased activity of mutant PAH.

Formation and cleavage of the iron-peroxypterin bridge.

Figure 4.

Two pathways differ in the proximity of the iron to the pterin cofactor and the H2O molecules assumed to be iron-coordinated during catalysis. According to one model, an iron dioxyge is formed as a resonance hybrid of Fe2+O2 and Fe3+O2-. The activated O2 attacks BH4, forming a charge separating electron-deficient pterin ring and electron-rich dioxygen species. Then Fe(II)-O-O-BH4 is formed. Meanwhile, assuming that BH4 is located in iron's first coordination shell and is not coordinated to H2O molecules. This mechanism involves a pterin radical and superoxide as critical intermediates. Then, Fe(II)-O-O-BH4 bridge is broken through  O-O bond to Fe(IV)=O and 4a-hydroxytetrahydrobiopterin.

Hydroxylation of phenylalanine by ferryl oxo intermediate

Figure 5.

Because the mechanism involves a Fe(IV)=O hydroxylating intermediate, oxidation of BH4 and hydroxylation of phenylalanine may result to H2O2 and unproductive BH4. If productive, Fe(IV)=O is added to Phe in an electrophilic aromatic substitution reaction, where iron is reduced from ferryl to ferrous sulfate. Tryptophan and tyrosine hydroxylases suggested that a cationic intermediate requires Fe(IV)=O to be with a water ligand. This undergoes a 1,2-hydride NIH shift, yielding a dienone intermediate to form a tyrosine. The carbinolamine product of PheOH was regenerated by a pterin cofactor to qBH2 that is reduced to BH4.

Associated Diseases of PheOH andTreatment:

Phenylketonuria

Any loss of PheH can result to neurotoxic metabolites of Phe. Also in depletion of tyrosine and methionine, the formation of cathecholamine neurotransmitters such as dopamine. Neurological disease is the outcome of such metabolic changes.

The entire length of protein was characterized under several mutations in the PheH. These resulted into a wide spectrum of phenotypes. Mild Hyperphenylalaninaemia(HPA) have ranged with a low impaired cognitive development to PKU, Phenylketonuria which is characterized by a high serum Phe, that causes severe progressive mental retardation, epilepsy and microcephaly if left untreated. In addition, it is said that 2% of cases arise from mutations in enzymes that synthesize BH4 cofactor.

Most of the mutations in PheH results to protein misfolding. Misfolded monomers tend to expose hydrophobic surfaces to form aberrant oligomers that form large protein aggregates. Cells remove mutant polypeptides to prevent damaging effects through ubiquitinylation. All forms of mutant enzyme, are subjected to increase rate of proteolytic degradation, where mutant PheH levels are reduced and Phe catabolism decreases to HPA or PKU diseases.

A small number of missense mutations for BH4 or substrate, results to a decreased enzyme affinity or affect allosteric regulatory properties of enzyme. For example, in the oral administration of BH4, some PheH deficiencies are responsive. Residual catalytic activity are shown in PheH deficiencies in response to BH4, but have reduced affinity. The administration of BH4 increased cofactor binding, as well as to protect from misfolding and degradation of mutant mutations by increasing protein stability.

Other genes may involve Phenylketonuria phenotype. For example, A brain injury caused by a toxic Phe metabolite occurs when a Phenylethylamine was degraded by a monoamine oxidase type 2 (MOAB) enzyme. MOAB is a modifier gene in Phenylketonuria, wherein similar levels of Phe have been identified in the different levels of phenylethylamine found in PKU patients.

Treatment for PheOH Disease

Treatment for patients with PKU varies depending on the levels of phenylalanine in the blood. An infant for example, with any form of PheOH deficiency should be evaluated immediately after birth to know whether the infant should be treated or not. A blood test can help in determining the amount of PheOH in the body, which will indicate the amount of Phe that the person could safely consume.

Patients with classic PKU must adhere a strict low-phenylalanine die. Other people with a mild form of the disease may take small amounts of amino acid.

In preserving a person's mental function with classic PKU requires a free form of Phe and a diet low in protein. Blood testing and diet should be done to maintain appropriate levels of phenylalanine. People with any form of PheOH should avoid taking aspartame, which contains phenylalanine.

Pregnant women with having a previous PheOH deficiency should maintain safe levels of phenylalanine to avoid passing birth defects on their children.

In the late 2007, Kuvan, a medication sapropterin dihydrochloride was approved by the FDA for people with PheOH deficiency to use. It can lower phenylalanine levels and enhance deficient enzyme in order to allow dietary restrictions to be relaxed. However, this treatment usually responds to people with a milder form of the disease.

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