Адам ағзасындағы фенилаланин гидроксилазасының құрылымы мен функциясы

Mammalian protein found in Homo sapiens

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.

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.

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.

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 Vmax or Km 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.

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. This model sets up the potential for an extensive investigation of PKU biology resembling PKU patients and for the evaluation of modern therapeutic strategies for PKU treatment.