Phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) () is an enzyme that catalyzes the hydroxylation of the aromatic side-chain of phenylalanine to generate tyrosine. PAH is one of three members of the biopterin-dependent aromatic amino acid hydroxylases, a class of monooxygenase that uses tetrahydrobiopterin (BH₄, a pteridine cofactor) and a non-heme iron for catalysis. During the reaction, molecular oxygen is heterolytically cleaved with sequential incorporation of one oxygen atom into BH₄ and phenylalanine substrate. In humans, mutations in its encoding gene, PAH, can lead to the metabolic disorder phenylketonuria.

{|

|thumb|alt=Reaction catalyzed by PAH.|Reaction catalyzed by PAH

|}

Enzyme mechanism

The reaction is thought to proceed through the following steps:

1. formation of a Fe(II)-O-O-BH₄ bridge.
2. heterolytic cleavage of the O-O bond to yield the ferryl oxo hydroxylating intermediate Fe(IV)=O
3. attack on Fe(IV)=O to hydroxylate phenylalanine substrate to tyrosine.

thumb|left|alt=Formation and cleavage of a Fe(II)-O-O-BH₄ bridge..|PAH mechanism, part I

Formation and cleavage of the iron-peroxypterin bridge. Although evidence strongly supports Fe(IV)=O as the hydroxylating intermediate, the mechanistic details underlying the formation of the Fe(II)-O-O-BH₄ bridge prior to heterolytic cleavage remain controversial. Two pathways have been proposed based on models that differ in the proximity of the iron to the pterin cofactor and the number of water molecules assumed to be iron-coordinated during catalysis. According to one model, an iron dioxygen complex is initially formed and stabilized as a resonance hybrid of Fe²⁺O₂ and Fe³⁺O₂⁻. The activated O₂ then attacks BH₄, forming a transition state characterized by charge separation between the electron-deficient pterin ring and the electron-rich dioxygen species. The Fe(II)-O-O-BH₄ bridge is subsequently formed. On the other hand, formation of this bridge has been modeled assuming that BH4 is located in iron's first coordination shell and that the iron is not coordinated to any water molecules. This model predicts a different mechanism involving a pterin radical and superoxide as critical intermediates. Once formed, the Fe(II)-O-O-BH₄ bridge is broken through heterolytic cleavage of the O-O bond to Fe(IV)=O and 4a-hydroxytetrahydrobiopterin; thus, molecular oxygen is the source of both oxygen atoms used to hydroxylate the pterin ring and phenylalanine.

thumb|left|alt=Hydroxylation of phenylalanine to tyrosine. |PAH mechanism, part II

Hydroxylation of phenylalanine by ferryl oxo intermediate. Because the mechanism involves a Fe(IV)=O (as opposed to a peroxypterin) hydroxylating intermediate, oxidation of the BH₄ cofactor and hydroxylation of phenylalanine can be decoupled, resulting in unproductive consumption of BH₄ and formation of H₂O₂. This cationic intermediate subsequently undergoes a 1,2-hydride NIH shift, yielding a dienone intermediate that then tautomerizes to form the tyrosine product. The pterin cofactor is regenerated by hydration of the carbinolamine product of PAH to quinonoid dihydrobiopterin (qBH₂), which is then reduced to BH₄.

Enzyme regulation

PAH is proposed to use the morpheein model of allosteric regulation.

Mammalian PAH exists in an equilibrium consisting of tetramers of two distinct architectures, with one or more dimeric forms as part of the equilibrium. This behavior is consistent with a dissociative allosteric mechanism. The resting-state form (RS-PAH) is architecturally distinct from the activated form (A-PAH). A full-length structure of A-PAH is currently lacking, but the Phe stabilized ACT-ACT interface that is characteristic of A-PAH has been determined and a structural model of A-PAH based on SAXS analysis has been proposed.

thumb|alt=Active site model for PAH.|Model of the active site of PAH bound to BH4, ferrous, and a phenylalanine analogue. (from PDB 1KW0) <span style="color:green;">Phenylalanine analogue</span>, <span style="color:blue;">BH4</span>, <span style="color:red;">iron</span>, <span style="color:tan;">Fe(II)-coordinated His and Glu residues</span>

Catalytic domain

Solved crystal structures of the catalytic domain indicate that the active site consists of an open and spacious pocket lined primarily by hydrophobic residues, though three glutamic acid residues, two histidines, and a tyrosine are also present and iron-binding. Inclusion of a Phe analogue in the crystal structure changes both iron from a six- to a five-coordinated state involving a single water molecule and bidentate coordination to Glu330 and opening a site for oxygen to bind. BH4 is concomitantly shifted toward the iron atom, although the pterin cofactor remains in the second coordination sphere. On the other hand, a competing model based on NMR and molecular modeling analyses suggests that all coordinated water molecules are forced out of the active site during the catalytic cycle while BH4 becomes directly coordinated to iron. As discussed above, resolving this discrepancy will be important for determining the exact mechanism of PAH catalysis.

N-terminal regulatory domain

The regulatory nature of the N-terminal domain (residues 1–117) is conferred by its structural flexibility. Hydrogen/deuterium exchanges analysis indicates that allosteric binding of Phe globally alters the conformation of PAH such that the active site is less occluded as the interface between the regulatory and catalytic domains is increasingly exposed to solvent. This observation is consistent with kinetic studies, which show an initially low rate of tyrosine formation for full-length PAH. This lag time is not observed, however, for a truncated PAH lacking the N-terminal domain or if the full-length enzyme is pre-incubated with Phe. Deletion of the N-terminal domain also eliminates the lag time while increasing the affinity for Phe by nearly two-fold; no difference is observed in the V_max or K_m for the tetrahydrobiopterin cofactor. Additional regulation is provided by Ser16; phosphorylation of this residue does not alter enzyme conformation but does reduce the concentration of Phe required for allosteric activation. Although both the homodimeric and homotetrameric forms of PAH are catalytically active, the two exhibit differential kinetics and regulation. In addition to reduced catalytic efficiency, the dimer does not display positive cooperativity toward L-Phe (which at high concentrations activates the enzyme), suggesting that L-Phe allosterically regulates PAH by influencing dimer-dimer interaction. Regulation of flux through phenylalanine-associated pathways is critical in mammalian metabolism, as evidenced by the toxicity of high plasma levels of this amino acid observed in phenylketonuria (see below). The principal source of phenylalanine is ingested proteins, but relatively little of this pool is used for protein synthesis. PAH is unusual among the aromatic amino acid hydroxylases for its involvement in catabolism; tyrosine and tryptophan hydroxylases, on the other hand, are primarily expressed in the central nervous system and catalyze rate-limiting steps in neurotransmitter/hormone biosynthesis. Mutations that have been identified in the PAH locus are documented at the Phenylalanine Hydroxylase Locus Knowledgbase (PAHdb, https://web.archive.org/web/20130718162051/http://www.pahdb.mcgill.ca/).

Since phenylketonuria can cause irreversible damage, it is imperative that deficiencies in the phenylalanine hydroxylase are determined early on in development. Originally, this was done using a bacterial inhibition assay known as the Guthrie Test. Now, PKU is part of newborn screening in many countries, and elevated phenylalanine levels are identified shortly after birth by measurement with tandem mass spectrometry. Placing the individual on a low phenylalanine, high tyrosine diet can help prevent any long-term damage to their development.

PAH Knock-out Model

The first attempt at creating a Pah-KO mouse model was reported in a research article published in 2021. This knockout mouse was created to be homozygous through its development within the C57BL/6 J strain using CRISPR/Cas9. Codon 7, GAG, in the Pah gene was altered to the stop codon TAG, depicting an intentional point mutation. Two to six-month-old, male, homozygous mice were studied by scientific methods such as behavioral and biochemical assays, MRI, and histopathology. The homozygous mice were routinely compared to control mice, age and sex-matched heterozygous Pah-KO mice that expressed sufficient PAH enzyme activity to supply phenylalanine and tyrosine levels similar to wild type mice.

The Pah-KO mouse model presented high blood phenylalanine and low tyrosine levels, hypocholesterolemia, high phenylalanine and low levels of neurotransmitters and tyrosine in the brain, hypomyelination, low brain and body weight, high grade of ocular pathology, hypopigmentation, and a progressive behavioral deficit compared to heterozygous mice. The increased amount of phenylalanine in whole brain homogenates of homozygous mice was similar to PKU patients. The hair coat color of both mice was dependent on the amount of melanin pigment in hair shafts; thus, homozygous mice were prone to be a lighter brown based on less melanin present. Mice behavior was tested with a nesting building assay.