Phenylalanine hydroxylase
Encyclopedia
Phenylalanine hydroxylase (PheOH, alternatively PheH or PAH) is an enzyme
that catalyzes the hydroxylation
of the aromatic side-chain of phenylalanine
to generate tyrosine
. PheOH is one of three members of the pterin-dependent 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.
Phenylalanine hydroxylase is the rate-limiting enzyme of the metabolic pathway
that degrades excess phenylalanine. Research on phenylalanine hydroxylase by Seymour Kaufman led to the discovery of tetrahydrobiopterin as a biological cofactor. The enzyme is also interesting from a human health perspective because mutations in PAH, the encoding gene, can lead to phenylketonuria, a severe metabolic disorder.
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.
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. When productive, though, the Fe(IV)=O intermediate is added to phenylalanine in an electrophilic aromatic substitution reaction that reduces iron from the ferryl to the ferrous state. Although initially an arene oxide or radical intermediate was proposed, analyses of the related tryptophan and tyrosine hydroxylases have suggested that the reaction instead proceeds through a cationic intermediate that requires Fe(IV)=O to be coordinated to a water ligand rather than a hydroxo group. 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 PheOH to quinonoid dihydrobiopterin (qBH2), which is then reduced to BH4.
In humans, this enzyme is expressed both in the liver and the kidney, and there is some indication that it may be differentially regulated in these tissues. PheOH 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.
(PKU) results. PKU is both genotypically and phenotypically heterogeneous: Over 300 distinct pathological mutants have been identified, the majority of which correspond to missense mutations that map to the catalytic domain. When a cohort of identified PheOH mutants were expressed in recombinant systems, the enzymes displayed altered kinetic behavior and/or reduced stability, consistent with structural mapping of these mutations to both the catalytic and tetramerization domains of the enzyme. Interestingly, BH44 has been administered as a pharmacological treatment and has been shown to reduce blood levels of phenylalanine for a segment of PKU patients whose genotypes lead to some residual PAH activity but have no defect in BH44 synthesis or regeneration. Follow-up studies suggest that in the case of certain PheOH mutants, excess BH44 acts as a pharmacological chaperone
to stabilize mutant enzymes with disrupted tetramer assembly and increased sensitivity to proteolytic cleavage and aggregation. Mutations that have been identified in the PAH locus are documented at the Phenylalanine Hydroxylase Locus Knowledgbase (PAHdb, http://www.pahdb.mcgill.ca/).
The three enzymes are homologous, that is, are thought to have evolved from the same ancient hydroxylase.
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
that catalyzes the hydroxylation
Hydroxylation
Hydroxylation is a chemical process that introduces a hydroxyl group into an organic compound. In biochemistry, hydroxylation reactions are often facilitated by enzymes called hydroxylases. Hydroxylation is the first step in the oxidative degradation of organic compounds in air...
of the aromatic side-chain of phenylalanine
Phenylalanine
Phenylalanine is an α-amino acid with the formula C6H5CH2CHCOOH. This essential amino acid is classified as nonpolar because of the hydrophobic nature of the benzyl side chain. L-Phenylalanine is an electrically neutral amino acid, one of the twenty common amino acids used to biochemically form...
to generate tyrosine
Tyrosine
Tyrosine or 4-hydroxyphenylalanine, is one of the 22 amino acids that are used by cells to synthesize proteins. Its codons are UAC and UAU. It is a non-essential amino acid with a polar side group...
. PheOH is one of three members of the pterin-dependent amino acid hydroxylases, a class of monooxygenase
Monooxygenase
Monooxygenases are enzymes that incorporate one hydroxyl group into substrates in many metabolic pathways. In this reaction, two atoms of dioxygen are reduced to one hydroxyl group and one H2O molecule by the concomitant oxidation of NADH.-Classification:...
that uses tetrahydrobiopterin
Tetrahydrobiopterin
Tetrahydrobiopterin or sapropterin is a naturally occurring essential cofactor of the three aromatic amino acid hydroxylase enzymes, used in the degradation of amino acid phenylalanine and in the biosynthesis of the neurotransmitters serotonin , melatonin, dopamine, norepinephrine ,...
(BH4, a pteridine
Pteridine
Pteridine is a chemical compound composed of fused pyrimidine and pyrazine rings. A pteridine is also a group of heterocyclic compounds containing a wide variety of substitutions on this structure. Pterins and flavins are classes of substituted pteridines that have important biological...
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.
Phenylalanine hydroxylase is the rate-limiting enzyme of the metabolic pathway
Metabolic pathway
In biochemistry, metabolic pathways are series of chemical reactions occurring within a cell. In each pathway, a principal chemical is modified by a series of chemical reactions. Enzymes catalyze these reactions, and often require dietary minerals, vitamins, and other cofactors in order to function...
that degrades excess phenylalanine. Research on phenylalanine hydroxylase by Seymour Kaufman led to the discovery of tetrahydrobiopterin as a biological cofactor. The enzyme is also interesting from a human health perspective because mutations in PAH, the encoding gene, can lead to phenylketonuria, a severe metabolic disorder.
Enzyme Mechanism
The reaction is thought to proceed through the following steps:- formation of a Fe(II)-O-O-BH4 bridge.
- heterolytic cleavage of the O-O bond to yield the ferryl oxo hydroxylating intermediate Fe(IV)=O
- attack on Fe(IV)=O to hydroxylate phenylalanine substrate to tyrosine.
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.
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. When productive, though, the Fe(IV)=O intermediate is added to phenylalanine in an electrophilic aromatic substitution reaction that reduces iron from the ferryl to the ferrous state. Although initially an arene oxide or radical intermediate was proposed, analyses of the related tryptophan and tyrosine hydroxylases have suggested that the reaction instead proceeds through a cationic intermediate that requires Fe(IV)=O to be coordinated to a water ligand rather than a hydroxo group. 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 PheOH to quinonoid dihydrobiopterin (qBH2), which is then reduced to BH4.
Structure
The PheOH monomer (51.9 kDa) consists of three distinct domains: a regulatory N-terminal domain (residues 1-117), the catalytic domain (residues 118-427), and a C-terminal domain (residues 428-453) responsible for oligomerization of identical monomers. Extensive crystallographic analysis has been performed, especially on the pterin- and iron-coordinated catalytic domain to examine the active site. The structure of the N-terminal regulatory domain has also been determined, and together with the solved structure of the homologous tyrosine hydroxylase C-terminal tetramerization domain, a structural model of tetrameric PheOH has been proposed.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 critical for pterin- and iron-binding. Contradictory evidence exists about the coordination state of the ferrous atom and its proximity to BH4 within the active site. According to crystallographic analysis, Fe(II) is coordinated by water, His285, His290, and Glu330 (a 2-his-1-carboxylate facial triad arrangement) with octahedral geometry. 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 concommitantly 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 PheOH 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 PheOH 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 PheOH. This lag time is not observed, however, for a truncated PheOH 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. This N-terminal regulatory domain is not observed in bacterial PheOHs but shows considerable structural homology to the regulatory domain of phosphogylcerate dehydrogenase, an enzyme in the serine biosynthetic pathway.Tetramerization domain
Prokaryotic PheOH is monomeric, whereas eukaryotic PheOH exists in an equilibrium between homotetrameric and homodimeric forms. The dimerization interface is composed of symmetry-related loops that link identical monomers, while the overlapping C-terminal tetramerization domain mediates the association of conformationally distinct dimers that are characterized by a different relative orientation of the catalytic and tetramerization domains (Flatmark, Erlandsen). The resulting distortion of the tetramer symmetry is evident in the differential surface area of the dimerization interfaces and distinguishes PheOH from the tetramerically symmetrical tyrosine hydroxylase. A domain-swapping mechanism has been proposed to mediate formation of the tetramer from dimers, in which C-terminal alpha-helixes mutually alter their conformation around a flexible C-terminal five-residue hinge region to form a coiled-coil structure, shifting equilibrium toward the tetrameric form. Although both the homodimeric and homotetrameric forms of PheOH 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 PheOH by influencing dimer-dimer interaction.Biological function
PheOH is a critical enzyme in phenylalanine metabolism and catalyzes the rate-limiting step in its complete catabolism to carbon dioxide and water. 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. Instead, the majority of ingested phenylalanine is catabolized through PheOH to form tyrosine; addition of the hydroxyl group allows for the benzene ring to be broken in subsequent catabolic steps. Transamination to phenylpyruvate, whose metabolites are excreted in the urine, represents another pathway of phenylalanine turnover, but catabolism through PheOH predominates.In humans, this enzyme is expressed both in the liver and the kidney, and there is some indication that it may be differentially regulated in these tissues. PheOH 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.
Disease relevance
Deficiency in PheOH activity due to mutations in the PAH gene causes hyperphenylalaninemia (HPA), and when blood phenylalanine levels increase above 20 times the normal concentration, the metabolic disease phenylketonuriaPhenylketonuria
Phenylketonuria is an autosomal recessive metabolic genetic disorder characterized by a mutation in the gene for the hepatic enzyme phenylalanine hydroxylase , rendering it nonfunctional. This enzyme is necessary to metabolize the amino acid phenylalanine to the amino acid tyrosine...
(PKU) results. PKU is both genotypically and phenotypically heterogeneous: Over 300 distinct pathological mutants have been identified, the majority of which correspond to missense mutations that map to the catalytic domain. When a cohort of identified PheOH mutants were expressed in recombinant systems, the enzymes displayed altered kinetic behavior and/or reduced stability, consistent with structural mapping of these mutations to both the catalytic and tetramerization domains of the enzyme. Interestingly, BH44 has been administered as a pharmacological treatment and has been shown to reduce blood levels of phenylalanine for a segment of PKU patients whose genotypes lead to some residual PAH activity but have no defect in BH44 synthesis or regeneration. Follow-up studies suggest that in the case of certain PheOH mutants, excess BH44 acts as a pharmacological chaperone
Pharmacological chaperone
A pharmacological chaperone is a small molecule that enters cells and serves as a molecular scaffolding in order to cause otherwise-misfolded mutant proteins to fold and route correctly within the cell....
to stabilize mutant enzymes with disrupted tetramer assembly and increased sensitivity to proteolytic cleavage and aggregation. Mutations that have been identified in the PAH locus are documented at the Phenylalanine Hydroxylase Locus Knowledgbase (PAHdb, http://www.pahdb.mcgill.ca/).
Related enzymes
Phenylalanine hydroxylase is closely related to two other enzymes:- tryptophan hydroxylaseTryptophan hydroxylaseTryptophan hydroxylase is an enzyme involved in the synthesis of the neurotransmitter serotonin. TPH catalyzes the following chemical reactionIt employs one cofactor, iron.- Function :...
(EC number 1.14.16.4), which controls levels of serotoninSerotoninSerotonin or 5-hydroxytryptamine is a monoamine neurotransmitter. Biochemically derived from tryptophan, serotonin is primarily found in the gastrointestinal tract, platelets, and in the central nervous system of animals including humans...
in the brain and the gastrointestinal tractGastrointestinal tractThe human gastrointestinal tract refers to the stomach and intestine, and sometimes to all the structures from the mouth to the anus. .... - tyrosine hydroxylaseTyrosine hydroxylaseTyrosine hydroxylase or tyrosine 3-monooxygenase is the enzyme responsible for catalyzing the conversion of the amino acid L-tyrosine to dihydroxyphenylalanine . It does so using tetrahydrobiopterin as a coenzyme. DOPA is a precursor for dopamine, which, in turn, is a precursor for norepinephrine ...
(EC number 1.14.16.2), which controls levels of dopamineDopamineDopamine is a catecholamine neurotransmitter present in a wide variety of animals, including both vertebrates and invertebrates. In the brain, this substituted phenethylamine functions as a neurotransmitter, activating the five known types of dopamine receptors—D1, D2, D3, D4, and D5—and their...
, epinephrineEpinephrineEpinephrine is a hormone and a neurotransmitter. It increases heart rate, constricts blood vessels, dilates air passages and participates in the fight-or-flight response of the sympathetic nervous system. In chemical terms, adrenaline is one of a group of monoamines called the catecholamines...
, and norepinephrineNorepinephrineNorepinephrine is the US name for noradrenaline , a catecholamine with multiple roles including as a hormone and a neurotransmitter...
in the brain and the adrenal medulla.
The three enzymes are homologous, that is, are thought to have evolved from the same ancient hydroxylase.