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Information on EC 1.14.16.1 - phenylalanine 4-monooxygenase and Organism(s) Rattus norvegicus and UniProt Accession P04176
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1.14.16.1 phenylalanine 4-monooxygenaseIUBMB Comments
The active centre contains mononuclear iron(II). The reaction involves an arene oxide that rearranges to give the phenolic hydroxy group. This results in the hydrogen at C-4 migrating to C-3 and in part being retained. This process is known as the NIH-shift. The 4a-hydroxytetrahydropteridine formed can dehydrate to 6,7-dihydropteridine, both spontaneously and by the action of EC 4.2.1.96, 4a-hydroxytetrahydrobiopterin dehydratase. The 6,7-dihydropteridine must be enzymically reduced back to tetrahydropteridine, by EC 1.5.1.34, 6,7-dihydropteridine reductase, before it slowly rearranges into the more stable but inactive compound 7,8-dihydropteridine.
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Word Map
- 1.14.16.1
- phenylketonuria
- hyperphenylalaninemia
- bh4
- error
- pterins
-
inborn
- children
- hydroxylases
-
neurotransmitter
-
province
- tetrahydropterins
-
counsel
-
intellectual
- dopamine
- l-tyrosine
-
genotype-phenotype
-
prenatal
- serotonin
- dihydropteridine
-
caucasian
- catecholamine
- hepatocytes
- sepiapterin
-
genotype-based
-
quinonoid
-
non-heme
-
chromobacterium
- neopterin
-
ligation-dependent
- dihydrochloride
-
neuropsychological
- lysolecithin
-
lifelong
- phenylpyruvate
- dopa
-
cyclohydrolase
- molecular biology
-
rflps
- p-chlorophenylalanine
- dihydrobiopterin
-
hypopigmentation
-
s-oxidation
- pteridine
- violaceum
The taxonomic range for the selected organisms is: Rattus norvegicus
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
Reaction Schemes
+
+
=
+
+
+
=
+
a 4a-hydroxy-5,6,7,8-tetrahydropteridine
Synonyms
phenylalanine hydroxylase, phenylalanine 4-monooxygenase, pheoh, phenylalanine 4-hydroxylase, phenylalanine monooxygenase, dicpah, cepah, l-phenylalanine 4-hydroxylase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
phenylalanine hydroxylase
-
oxygenase, phenylalanine 4-mono-
-
-
-
-
PAH
phenylalaninase
-
-
-
-
phenylalanine 4-hydroxylase
-
-
-
-
phenylalanine hydroxylase
PAH
-
-
-
-
phenylalanine hydroxylase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
L-phenylalanine + a 5,6,7,8-tetrahydropteridine + O2 = L-tyrosine + a 4a-hydroxy-5,6,7,8-tetrahydropteridine
hydrogen/deuterium exchange monitored by mass spectrometry is used to gain insight into local conformational changes accompanying activation of rat phenylalanine hydroxylase by phenylalanine. Peptides in the regulatory and catalytic domains that lie in the interface between these two domains show large increases in the extent of deuterium incorporation from solvent in the presence of phenylalanine
L-phenylalanine + a 5,6,7,8-tetrahydropteridine + O2 = L-tyrosine + a 4a-hydroxy-5,6,7,8-tetrahydropteridine
L-phenylalanine + a 5,6,7,8-tetrahydropteridine + O2 = L-tyrosine + a 4a-hydroxy-5,6,7,8-tetrahydropteridine
catalytic mechanism
-
L-phenylalanine + a 5,6,7,8-tetrahydropteridine + O2 = L-tyrosine + a 4a-hydroxy-5,6,7,8-tetrahydropteridine
substrate and oxygen binding site, reaction mechanism, structure-function analysis
-
L-phenylalanine + a 5,6,7,8-tetrahydropteridine + O2 = L-tyrosine + a 4a-hydroxy-5,6,7,8-tetrahydropteridine
1H-15N HSQC NMR spectra are obtained of the 15N-labeled protein alone and in the presence of phenylalanine. Regulatory domain of phenylalanine hydroxylase can bind phenylalanine, consistent with the presence of an allosteric site for the amino acid
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
-
oxidation
-
-
-
-
reduction
-
-
-
-
PATHWAY SOURCE
PATHWAYS
BRENDA
KEGG
-
-, -, -
SYSTEMATIC NAME
IUBMB Comments
L-phenylalanine,tetrahydrobiopterin:oxygen oxidoreductase (4-hydroxylating)
The active centre contains mononuclear iron(II). The reaction involves an arene oxide that rearranges to give the phenolic hydroxy group. This results in the hydrogen at C-4 migrating to C-3 and in part being retained. This process is known as the NIH-shift. The 4a-hydroxytetrahydropteridine formed can dehydrate to 6,7-dihydropteridine, both spontaneously and by the action of EC 4.2.1.96, 4a-hydroxytetrahydrobiopterin dehydratase. The 6,7-dihydropteridine must be enzymically reduced back to tetrahydropteridine, by EC 1.5.1.34, 6,7-dihydropteridine reductase, before it slowly rearranges into the more stable but inactive compound 7,8-dihydropteridine.
CAS REGISTRY NUMBER
COMMENTARY
9029-73-6
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + 4a-hydroxytetrahydrobiopterin
-
-
-
?
4-methylphenylalanine + 6,7-dimethyl-tetrahydropterin + O2
4-(hydroxymethyl)phenylalanine + 3-methyltyrosine + H2O + 6,7-dimethyl-dihydropterin
-
-
79% methyl-hydroxylation, 21% para-hydroxylation, shift of para-substituent by NIH shift mechanism
?
L-cyclohexylalanine + 6,7-dimethyl-tetrahydropterin + O2
4-hydroxy-L-cyclohexylalanine + H2O + 6,7-dimethyl-dihydropterin
-
50% less active than the enzyme from Chromobacterium violaceum
-
?
L-methionine + tetrahydrobiopterin + O2
?
-
lysolecithin activated enzyme
-
-
?
L-norleucine + tetrahydrobiopterin + O2
?
-
lysolecithin activated enzyme
-
-
?
L-phenylalanine + (6R)-tetrahydrobiopterin + O2
L-tyrosine + (6R)-dihydrobiopterin + H2O
-
in mammals rate-limiting step in complete catabolism of phenylalanine to CO2 and water
-
?
L-phenylalanine + 5,6,7,8-tetrahydrobiopterin + O2
L-tyrosine + 4a-hydroxy-tetrahydrobiopterin
L-phenylalanine + 5,6,7,8-tetrahydrobiopterin + O2
L-tyrosine + 4a-hydroxytetrahydrobiopterin
-
-
-
-
?
L-phenylalanine + 6,7-dimethyltetrahydrobiopterin
L-tyrosine + 6,7-dimethyl-4a-hydroxy-tetrahydrobiopterin
-
-
-
-
r
L-phenylalanine + 6-methyl-tetrahydrobiopterin + O2
L-tyrosine + 6-methyl-4-hydroxy-tetrahydrobiopterin
-
-
-
-
?
L-phenylalanine + 6-methyltetrahydrobiopterin + O2
L-tyrosine + 6-methyl-4a-hydroxytetrahydrobiopterin
-
-
-
-
?
L-phenylalanine + 6-methyltetrahydropterin + O2
L-tyrosine + 4a-hydroxy-6-methyltetrahydropterin
-
low activity with 6-methyltetrahydropterin
-
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + 4a-hydroxy-tetrahydrobiopterin
-
-
-
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + 4a-hydroxytetrahydrobiopterin
-
-
-
-
?
S-carboxy-methyl-L-cysteine + 5,6,7,8-tetrahydrobiopterin + O2
S-carboxymethyl-L-cysteine S-oxide + dihydrobiopterin + H2O
-
poor substrate
-
-
?
S-carboxymethyl-L-cysteine + tetrahydrobiopterin + O2
S-carboxymethyl-L-cysteine S-oxide + dihydrobiopterin + H2O
-
-
-
-
?
S-methyl-L-cysteine + 5,6,7,8-tetrahydrobiopterin + O2
S-methyl-L-cysteine S-oxide + dihydrobiopterin + H2O
-
poor substrate
-
-
?
S-methyl-L-cysteine + tetrahydrobiopterin + O2
?
-
lysolecithin activated enzyme
-
-
?
S-methyl-L-cysteine + tetrahydrobiopterin + O2
S-methyl-L-cysteine S-oxide + dihydrobiopterin + H2O
-
-
-
-
?
L-phenylalanine + 5,6,7,8-tetrahydrobiopterin + O2
L-tyrosine + 4a-hydroxy-tetrahydrobiopterin
-
-
-
-
?
L-phenylalanine + 5,6,7,8-tetrahydrobiopterin + O2
L-tyrosine + 4a-hydroxy-tetrahydrobiopterin
-
specific for the L-isomer, no activity with the D-isomer, effects of isotopic substrates [4-2H]-, [3,5-2H2]-, and 2H5-phenylalanine, overview
-
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
-
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
-
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
-
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
-
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
-
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
-
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
-
-
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
relatively low activity with tetrahydrobiopterin can be selectively increased by limited proteolysis, alkylation of sulfhydryl groups with N-ethylmaleimide or phosphorylation by cAMP-dependent protein kinase
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
relatively low activity with tetrahydrobiopterin can be selectively increased by limited proteolysis
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
relatively low activity with tetrahydrobiopterin can be selectively increased by limited proteolysis
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
relatively low activity with tetrahydrobiopterin can be selectively increased by limited proteolysis
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
additional electron donors: 6-methylpterin, 6,7-dimethyltetrahydropterin
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
additional electron donors: 6-methylpterin, 6,7-dimethyltetrahydropterin
4-alpha-carbinolamine is the first free pterin product formed
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
specificity is quantitatively altered when the enzyme is activated by lysolecithin
4-alpha-carbinolamine is the first free pterin product formed
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
additional electron donors: 6-methyltetrahydropterin, 7-methylpterin, and 2,4,5-triamino-6-hydroxypyrimidine
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
additional electron donors: 6-methyltetrahydropterin, 7-methylpterin, and 2,4,5-triamino-6-hydroxypyrimidine
4-alpha-carbinolamine is the first free pterin product formed
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
non activated enzyme has much greater activity with 6-methyltetrahydropterin and dimethyltetrahydropterin than with tetrahydrobiopterin
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
low activity with tetrahydrobiopterin can be selectively increased by a wide variety of reversible and irreversible modificators of the enzyme, e.g. interaction with phospholipids
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
low activity with tetrahydrobiopterin can be selectively increased by a wide variety of reversible and irreversible modificators of the enzyme, e.g. interaction with phospholipids
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
low activity with tetrahydrobiopterin can be selectively increased by a wide variety of reversible and irreversible modificators of the enzyme, e.g. interaction with phospholipids
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
low activity with tetrahydrobiopterin can be selectively increased by a wide variety of reversible and irreversible modificators of the enzyme, e.g. interaction with phospholipids
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + dihydrobiopterin + H2O
-
low activity with tetrahydrobiopterin can be selectively increased by a wide variety of reversible and irreversible modificators of the enzyme, e.g. interaction with phospholipids
4-alpha-carbinolamine is the first free pterin product formed
?
additional information
?
-
-
the enzyme is important in the metabolism of xenobiotic thioether substrates, overview
-
-
?
additional information
?
-
-
the rate of clearance of the substrates via rat PAH is L-phenylalanine > L-methionine > S-carboxy-methyl-L-cysteine > S-methyl-L-cysteine
-
-
?
additional information
?
-
-
thiodiglycolic acid is not a substrate for PAH
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + 4a-hydroxytetrahydrobiopterin
-
-
-
?
L-phenylalanine + (6R)-tetrahydrobiopterin + O2
L-tyrosine + (6R)-dihydrobiopterin + H2O
-
in mammals rate-limiting step in complete catabolism of phenylalanine to CO2 and water
-
?
L-phenylalanine + 5,6,7,8-tetrahydrobiopterin + O2
L-tyrosine + 4a-hydroxy-tetrahydrobiopterin
-
-
-
-
?
L-phenylalanine + tetrahydrobiopterin + O2
L-tyrosine + 4a-hydroxytetrahydrobiopterin
-
-
-
-
?
additional information
?
-
-
the enzyme is important in the metabolism of xenobiotic thioether substrates, overview
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
tetrahydrobiopterin
-
-
438654, 438655, 438714, 438715, 438716, 438721, 438722, 438725, 438726, 438729, 438731, 684717, 688016
additional information
-
artificial cofactors: 6-methyltetrahydropterin, 6,7-dimethyltetrahydropterin
-
additional information
-
artificial cofactors: 6-methyltetrahydropterin, 6,7-dimethyltetrahydropterin
-
additional information
-
artificial cofactor: 2,4,5-triamino-6-hydroxypyrimidine
-
additional information
-
artificial cofactors: 6-methyltetrahydropterin, 7-methylpterin
-
additional information
-
artificial cofactors: 6-methyltetrahydropterin, 7-methylpterin
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
Iron
Fe2+
-
nonheme iron is bound on one face by residues His285, His290, and Glu330 forming a a 2-His-1-carboxylate facial triad, the three ligands differ in their sensitivity to mutagenesis, structure, overview
Iron
-
1 iron atom per subunit, fully active enzyme has non-heme high-spin ferric iron coordinated at the active site
Iron
-
iron can be removed by treatment with o-phenanthroline, reconstitution of apoenzyme with iron restores 90% of the initial activity
Iron
-
kinetic data suggest, that enzyme's iron is solvent-accessible and resides in a hydrophobic pocket of the enzyme
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2,3-dihydroxynaphthalene
-
binds to Fe3+ on enzyme that is oxidized during catalysis
3,4-Dihydroxystyrene
-
0.0005-0.005 mM, 50% inhibition, noncompetitive vs. 6,7-dimethyltetrahydropterin and L-phenylalanine
Acetohydroxamate
-
competitive vs. tetrahydrobiopterin, most probably due to chelation of enzyme's iron
bathophenanthroline
-
competitive vs. 6-methyl-5,6,7,8-tetrahydropterin and tetrahydrobiopterin, most probably due to chelation of enzyme's iron
benzohydroxamate
-
competitive vs. tetrahydrobiopterin, most probably due to chelation of enzyme's iron
S-carboxy-methyl-L-cysteine
-
competitive inhibitor of the C-oxidation of L-phenylalanine
S-carboxymethyl-L-cysteine
-
competitive to L-phenylalanine, 5 mM, 74% residual activity with substrate L-phenylalanine
S-methyl-ergothionine
-
competitive inhibitor of the C-oxidation of L-phenylalanine
(6R)-L-erythro-tetrahydrobiopterin
-
inhibits phenylalanine and lysolecithin activation
dithiothreitol
-
2 mM, 94% inhibition, dithiothreitol induces the dissociation of iron from the enzyme, catalase and phenylalanine partially protect from inhibition
S-methyl-L-cysteine
-
competitive to L-phenylalanine, 5 mM, 89% residual activity with substrate L-phenylalanine
S-methyl-L-cysteine
-
competitive inhibitor of the C-oxidation of L-phenylalanine
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
alpha-chymotrypsin
-
limited proteolysis of purified liver enzyme, 20-30fold increase in activity, cofactor tetrahydrobiopterin
-
cAMP-dependent protein kinase
-
2-4fold increase in activity in the presence of tetrahydrobiopterin
-
lysolecithin
-
1 mM, approx. 7.5fold stimulation of phosphorylated recombinant wild-type enzyme, S16E and S16D mutant enzymes, approx. 24fold stimulation of non-phosphorylated recombinant wild-type enzyme, S16A, S16Q, S16N and S16K mutant enzymes
phenylalanine
-
major regulator of liver enzyme, binds at an effector site converting the inactive to an active enzyme
phenylalanine
-
homotropic allosteric activator of both hepatic and recombinant enzymes
phenylalanine
-
binds to activation site distinct from catalytic site with a stoichiometry of 1/enzyme subunit, binding induces a conformation change converting the enzyme tetramer from an inactive to an active form, activation site binds phenylalanine in a cooperative manner, regulation of activation
Phospholipids
-
increase in activity in the presence of tetrahydrobiopterin but not in the presence of synthetic pterin cofactors
additional information
not significantly activated by L-valine, L-alanine, and L-serine
-
additional information
-
relatively low activity with tetrahydrobiopterin can be selectively increased by limited proteolysis
-
additional information
-
relatively low activity with tetrahydrobiopterin can be selectively increased by limited proteolysis
-
additional information
-
relatively low activity with tetrahydrobiopterin can be selectively increased by limited proteolysis
-
additional information
-
relatively low activity with tetrahydrobiopterin can be selectively increased by phosphorylation
-
additional information
-
non activated enzyme has much greater activity with 6-methyltetrahydropterin and dimethyltetrahydropterin than with tetrahydrobiopterin
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
55.97
N-acetyl-S-carboxymethyl-L-cysteine
-
wild type enzyme from hepatic cytosol
14.73
S-carboxy-methyl-L-cysteine
-
at 37°C, 50 mM potassium phosphate buffer, pH 6.8
0.45
S-methyl-ergothionine
-
at 37°C, 50 mM potassium phosphate buffer, pH 6.8
0.0344
6,7-dimethyl-5,6,7,8-tetrahydropterin
-
enzyme form II, substrate L-phenylalanine
0.0444
6,7-dimethyl-5,6,7,8-tetrahydropterin
-
enzyme form I, substrate L-phenylalanine
0.105
6,7-dimethyltetrahydrobiopterin
-
chymotrypsin activated liver enzyme
0.105
6,7-dimethyltetrahydrobiopterin
-
chymotrypsin-activated enzyme, at pH 6.8 and 25°C
0.037
6-Methyl-5,6,7,8-tetrahydropterin
-
enzyme form I, substrate L-phenylalanine
0.0455
6-Methyl-5,6,7,8-tetrahydropterin
-
enzyme form II, substrate L-phenylalanine
0.063
6-methyl-tetrahydrobiopterin
-
pH 7.0, 25°C, recombinant mutant DELTA 117PheH V379D
0.083
6-methyl-tetrahydrobiopterin
-
pH 7.0, 25°C, recombinant mutant DELTA 117PheH
0.12
L-phenylalanine
-
pH 7.0, 25°C, with cofactor 6-methyl-tetrahydrobiopterin, recombinant mutant DELTA 117PheH
0.42
L-phenylalanine
-
pH 7.0, 25°C, with cofactor 6-methyl-tetrahydrobiopterin, recombinant mutant DELTA 117PheH V379D
0.454
L-phenylalanine
-
cofactor 6-methyl-5,6,7,8-tetrahydropterin, enzyme form II
0.72
L-phenylalanine
-
cofactor 6-methyl-5,6,7,8-tetrahydropterin, enzyme form I
1.43
L-phenylalanine
-
cofactor 6,7-dimethyl-5,6,7,8-tetrahydropterin, enzyme form II
1.92
L-phenylalanine
-
cofactor 6,7-dimethyl-5,6,7,8-tetrahydropterin, enzyme form I
8.5
L-tryptophan
-
cofactor 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine, enzyme form I
43.25
S-methyl-L-cysteine
-
at 37°C, 50 mM potassium phosphate buffer, pH 6.8
1.7
thienylalanine
-
cofactor tetrahydrobiopterin, lysolecithin activated enzyme
additional information
additional information
-
binding constants for Fe2+ of wild-type and mutant enzymes, overview
-
additional information
additional information
-
kinetic and molecular modelling of sulfur-containing substrates, overview
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.031 - 0.51
6-methyl-tetrahydrobiopterin
-
pH 7.0, 25°C, recombinant mutant DELTA 117PheH
1.12
6-methyl-tetrahydrobiopterin
-
pH 7.0, 25°C, recombinant mutant DELTA 117PheH V379D
7.85
6-methyl-tetrahydrobiopterin
-
pH 7.0, 25°C, recombinant mutant DELTA 117PheH
0.04 - 1.97
L-phenylalanine
-
pH 7.0, 25°C, with cofactor 6-methyl-tetrahydrobiopterin, recombinant mutant DELTA 117PheH
0.3
L-phenylalanine
-
pH 7.0, 25°C, with cofactor 6-methyl-tetrahydrobiopterin, recombinant mutant DELTA 117PheH V379D
8.55
L-phenylalanine
-
pH 7.0, 25°C, with cofactor 6-methyl-tetrahydrobiopterin, recombinant mutant DELTA 117PheH
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
13.14
S-carboxy-methyl-L-cysteine
-
at 37°C, 50 mM potassium phosphate buffer, pH 6.8
0.5
S-methyl-ergothionine
-
at 37°C, 50 mM potassium phosphate buffer, pH 6.8
41.53
S-methyl-L-cysteine
-
at 37°C, 50 mM potassium phosphate buffer, pH 6.8
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
1.46
5
-
phosphorylated recombinant wild-type enzyme, S16N and S16D mutant enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
phenylalanine hydroxylase dysfunction causes phenylketonuria
physiological function
-
key enzyme in the sulfoxidation of S-carboxymethyl-L-cysteine S-oxide and its thioester metabolites S-methyl-L-cysteine, N-acetyl-S-carboxymethyl-L-cysteine, and N-acetyl-S-methyl-L-cysteine
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PH4H_RAT
453
0
51822
Swiss-Prot
other Location (Reliability: 2)
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
50000
100000 - 110000
13000
-
2 * 13000 (regulatory domain 1-118), gel filtration. In the presence of phenylalanine, the protein elutes earlier from the column, consistent with a conformational change in the presence of the amino acid
200000 - 210000
51000 - 55000
-
monomeric form, enzyme exists as monomer, dimer and tetramer
100000 - 110000
-
dimeric form, enzyme exists as monomer, dimer and tetramer
200000 - 210000
-
tetrameric species accounts for 78% of the total enzyme, enzyme exists as monomer, dimer and tetramer
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homotetramer
dimer
tetramer
additional information
dimer
-
2 * 13000 (regulatory domain 1-118), gel filtration. In the presence of phenylalanine, the protein elutes earlier from the column, consistent with a conformational change in the presence of the amino acid
additional information
-
some authors report that the enzyme exists as a mixture of dimers and tetramers, others report that it exists solely as tetramer or as dimer, percentage of dimers increases on frozen storage, preincubation of the enzyme with phenylalanine leads to even higher than 200000 Da molecular weight forms
additional information
-
rat liver enzyme and recombinant human enzyme are in a tetramer/dimer equilibrium
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
side-chain modification
additional information
-
hepatic enzyme consists of a mixture of phosphorylated and nonphosphorylated forms
side-chain modification
-
liver enzyme in untreated animals is a mixture of phosphorylated and nonphosphorylated forms, enzyme may be phosphorylated in vivo by cAMP-dependent protein kinase
side-chain modification
-
recombinant enzyme incorporates 0.97 phosphate per subunit
side-chain modification
-
enzyme contains 0.3 mol of phosphate per mol of enzyme subunit
side-chain modification
-
enzyme form I contains 0.3 mol phosphate per mol subunit, enzyme form II contains 0.4 mol
side-chain modification
-
phosphorylated enzyme shows a 3.3fold increase in Vmax
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
sitting drop vapor diffusion method, using 0.1 M Bis-Tris-HCl pH 5.5, 25% (w/v) polyethylene glycol 3350, and 0.2 M magnesium chloride
vapor diffusion method, using 140 mM Na-acetate, 70 mM Na-citrate, 100 mM Na-cacodylate (pH 6.5), and 31.5% (w/v) PEG 1000
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A47G
the mutation decreases the affinity of the catalytic domain for L-phenylalanine compared to the wild type
DELTA1-117
mutant lacking the first 117 amino acids containing only the catalytic and tetramerization domains: the effects of phenylalanine on the hydrogen/deuterium exchange kinetics are limited to peptides surrounding the binding site for the amino acid substrate
E44Q
the mutation decreases the affinity of the catalytic domain for L-phenylalanine compared to the wild type and leads to loss of activity
H64N
the mutation decreases the affinity of the catalytic domain for L-phenylalanine compared to the wild type
L48V
the mutation decreases the affinity of the catalytic domain for L-phenylalanine compared to the wild type
L62V
the mutation decreases the affinity of the catalytic domain for L-phenylalanine compared to the wild type and leads to loss of activity
A322S/V379D
-
truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity
E280A
-
site-directed mutagenesis of catalytic core mutant DELTA117PheH, 70% reduced activity but unaltered isotopic effects of isotope substrates
E330H
-
site-directed mutagenesis of a metal ligand binding residue, the mutant enzyme shows over 80% reduced activity compared to the wild-type enzyme
E330Q
-
site-directed mutagenesis of a metal ligand binding residue, the mutant enzyme shows over 80% reduced activity compared to the wild-type enzyme
F263A
-
site-directed mutagenesis of catalytic core mutant DELTA117PheH, 85% reduced activity but unaltered isotopic effects of isotope substrates
H264Q/V379D
H264Q/Y277H/V379D
H285E
-
site-directed mutagenesis of a metal ligand binding residue, the mutant enzyme shows over 80% reduced activity compared to the wild-type enzyme
H285Q
-
site-directed mutagenesis of a metal ligand binding residue, the mutant enzyme shows 80% reduced activity compared to the wild-type enzyme
H290E
-
site-directed mutagenesis of a metal ligand binding residue, the mutant enzyme shows over 80% reduced activity compared to the wild-type enzyme
H290Q
-
site-directed mutagenesis of a metal ligand binding residue, the mutant enzyme shows over 80% reduced activity compared to the wild-type enzyme
L293M
-
truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity
R270K
-
the mutation effectively abolishes binding of L-phenylalanine in the active site and leads to strongly reduced kcat/Km value (10000 down) compared to the wild type enzyme
S16E
-
slightly higher Km for tetrahydrobiopterin than wild-type, approx. 3fold higher Vmax with phenylalanine
S16N
-
slightly higher Km for tetrahydrobiopterin than wild-type, approx. 3fold higher Vmax with phenylalanine
S16Q
-
slightly higher Km for tetrahydrobiopterin than wild-type, similar Vmax with phenylalanine
S251A
-
truncated enzyme containing the catalytic domain, no tyrosine hydroxylation activity
S251A/H264Q
-
truncated enzyme containing the catalytic domain, no tyrosine hydroxylation activity
S251A/H264Q/V379D
-
truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity
S251A/H264Q/Y277H
-
truncated enzyme containing the catalytic domain, no tyrosine hydroxylation activity
S251A/H264Q/Y277H/A322S
-
truncated enzyme containing the catalytic domain, no tyrosine hydroxylation activity
S251A/H264Q/Y277H/A322S/V379D
-
truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity
S251A/H264Q/Y277H/A322S/V379D/Y356H
-
truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity
S251A/H264Q/Y277H/A322S/V379D/Y356H/L293M
-
truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity
S251A/H264Q/Y277H/V379D
-
truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity
S251A/V379D
-
truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity
V379D
Y277H/V379D
-
truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity
additional information
-
construction and catalytic properties of deletion mutant DELTA117PheH, consisting of the catalytic core of the enzyme, the isotopic effects of substrates [4-2H]-, [3,5-2H2]-, and 2H5-phenylalanine are unaltered compared to the wild-type enzyme, overview
H264Q/V379D
-
truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity
H264Q/V379D
-
double mutant of full length enzyme, shows significant tyrosine hydroxylation activity
H264Q/Y277H/V379D
-
truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity
H264Q/Y277H/V379D
-
triple mutant of full length enzyme, shows significant tyrosine hydroxylation activity
V379D
-
truncated enzyme containing the catalytic domain, mutant shows tyrosine hydroxylation activity
V379D
-
site-directed mutagenesis of catalytic core mutant DELTA117PheH, the isotopic effects of substrates [4-2H]-, [3,5-2H2]-, and 2H5-phenylalanine are altered compared to the wild-type enzyme, overview
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
phenyl Sepharose column chromatography and HiTrap Q column chromatography
affinity chromatography on 6,7-dimethyl-5,6,7,8-tetrahydropterin-Sepharose 4B
-
distinct forms I and II differing in phosphate content and isoelectric point
-
phenyl Sepharose column chromatography and Q Sepharose column chromatography, and Superdex 200 gel filtration
-
recombinant catalytic core mutant DELTA 117PheH and point mutants thereof from Escherichia coli strain C41(DE3)
-
recombinant wild-type S16E, S16Q, S16N, S16D, S16A, and S16K mutant enzyme
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of catalytic core mutant DELTA 117PheH and point mutants thereof in Escherichia coli strain C41(DE3)
-
expression of truncated enzyme containing the catalytic domain and various mutants in Escherichia coli
-
expression of wild-type and S16E, S16Q, S16N, S16D, S16A, and S16K mutant enzyme in Escherichia coli
-
regulatory domain (amino acids 1-118) of rat phenylalanine hydroxylase is expressed in Escherichia coli
-
the truncated enzyme PheHDELTA117 is expressed in Escherichia coli BL21(DE3) cells
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Kaufman, S.
Aromatic amino acid hydroxylases
The Enzymes, 3rd Ed. (Boyer, P. D. , ed. )
18
217-282
1987
Homo sapiens, Rattus norvegicus
-
Koizumi, S.; Matsushima, Y.; Nagatsu, T.; Linuma, H.; Takeuchi, T.; Umezawa, H.
3,4-Dihydroxystyrene, a novel microbial inhibitor for phenylalanine hydroxylase and other pteridine-dependent monooxygenases
Biochim. Biophys. Acta
789
111-118
1984
Rattus norvegicus
Daubner, S.C.; Melendez, J.; Fitzpatrick, P.F.
Reversing the substrate specificities of phenylalanine and tyrosine hydroxylase: aspartate 425 of tyrosine hydroxylase is essential for L-DOPA formation
Biochemistry
39
9652-9661
2000
Rattus norvegicus
Kaufman, S.
Phenylalanine 4-monooxygenase from rat liver
Methods Enzymol.
142
3-17
1987
Rattus norvegicus
Shiman, R.
Purification and assay of rat liver phenylalanine 4-monooxygenase
Methods Enzymol.
142
17-27
1987
Rattus norvegicus
Abita, J.P.; Blandin-Savoja, F.; Rey, F.
Phenylalanine 4-monooxygenase from human liver
Methods Enzymol.
142
27-35
1987
Homo sapiens, Rattus norvegicus
Nakata, H.; Fujisawa, H.
Purification and characterization of phenylalanine 4-monooxygenase from rat liver
Biochim. Biophys. Acta
614
313-327
1980
Rattus norvegicus
Al-Janabi, J.M.
Purification of rat liver phenylalanine hydroxylase by affinity chromatography
Arch. Biochem. Biophys.
200
603-608
1980
Rattus norvegicus
Martinez, A.; Andersson, K.K.; Haavik, J.; Flatmark, T.
EPR and 1H-NMR spectroscopic studies on the paramagnetic iron at the active site of phenylalanine hydroxylase and its interaction with substrates and inhibitors
Eur. J. Biochem.
198
675-682
1991
Bos taurus, Rattus norvegicus
Martinez, A.; Olafsdottir, S.; Haavik, J.; Flatmark, T.
Inactivation of purified phenylalanine hydroxylase by dithiothreitol
Biochem. Biophys. Res. Commun.
182
92-98
1992
Rattus norvegicus
Bloom, L.M.; Benkovic, S.J.; Gaffney, B.J.
Characterization of phenylalanine hydroxylase
Biochemistry
25
4204-4210
1986
Rattus norvegicus
Abita, J.P.; Parniak, M.; Kaufman, S.
The activation of rat liver phenylalanine hydroxylase by limited proteolysis, lysolecithin, and tocopherol phosphate. Changes in conformation and catalytic properties
J. Biol. Chem.
259
14560-14566
1984
Rattus norvegicus
Shiman, R.; Gray, D.W.; Hill, M.A.
Regulation of rat liver phenylalanine hydroxylase. I. Kinetic properties of the enzyme's iron and enzyme reduction site
J. Biol. Chem.
269
24637-24646
1994
Rattus norvegicus
Shiman, R.; Xia, T.; Hill, M.A.; Gray, D.W.
Regulation of rat liver phenylalanine hydroxylase. II. Substrate binding and the role of activation in the control of enzymic activity
J. Biol. Chem.
269
24647-24656
1994
Rattus norvegicus
Kowlessur, D.; Yang, X.J.; Kaufman, S.
Further studies of the role of Ser-16 in the regulation of the activity of phenylalanine hydroxylase
Proc. Natl. Acad. Sci. USA
92
4743-4747
1995
Rattus norvegicus
Kappock, T.J.; Harkins, P.C.; Friedenberg, S.; Caradonna, J.P.
Spectroscopic and kinetic properties of unphosphorylated rat hepatic phenylalanine hydroxylase expressed in Escherichia coli. Comparison of resting and activated states
J. Biol. Chem.
270
30532-30544
1995
Rattus norvegicus
Carr, R.T.; Balasubramanian, S.; Hawkins, P.C.D.; Benkovic, S.J.
Mechanism of metal-independent hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase
Biochemistry
34
7525-7532
1995
Chromobacterium violaceum, Rattus norvegicus
Kowlessur, D.; Citron, B.A.; Kaufman, S.
Recombinant human phenylalanine hydroxylase: novel regulatory and structural properties
Arch. Biochem. Biophys.
333
85-95
1996
Homo sapiens, Rattus norvegicus
Bjorgo, E.; De Carvalho, R.M.N.; Flatmark, T.
A comparison of kinetic and regulatory properties of the tetrameric and dimeric forms of wild-type and Thr427->Pro mutant human phenylalanine hydroxylase. Contribution of the flexible hinge region Asp425-Gln429 to the tetramerization and cooperative substrate binding
Eur. J. Biochem.
268
997-1005
2001
Homo sapiens, Rattus norvegicus
Goreish, A.H.; Bednar, S.; Jones, H.; Mitchell, S.C.; Steventon, G.B.
Phenylalanine 4-monooxygenase and the S-oxidation of S-carboxymethyl-L-cysteine
Drug Metabol. Drug Interact.
20
159-174
2004
Rattus norvegicus, Rattus norvegicus Wistar
Pavon, J.A.; Fitzpatrick, P.F.
Insights into the catalytic mechanisms of phenylalanine and tryptophan hydroxylase from kinetic isotope effects on aromatic hydroxylation
Biochemistry
45
11030-11037
2006
Rattus norvegicus
Teigen, K.; Jensen, V.R.; Martinez, A.
The reaction mechanism of phenylalanine hydroxylase. - A question of coordination
Pteridines
16
27-34
2005
Chromobacterium violaceum, Homo sapiens, Rattus norvegicus
-
Li, J.; Fitzpatrick, P.F.
Characterization of metal ligand mutants of phenylalanine hydroxylase: Insights into the plasticity of a 2-histidine-1-carboxylate triad
Arch. Biochem. Biophys.
475
164-168
2008
Rattus norvegicus
Patel, N.G.; Iliadou, C.; Boonyapiwat, B.; Barlow, D.J.; Forbes, B.; Mitchell, S.C.; Steventon, G.B.
Enzyme kinetic and molecular modelling studies of sulphur-containing substrates of phenylalanine 4-monooxygenase
J. Enzyme Inhib. Med. Chem.
23
958-63
2007
Homo sapiens, Rattus norvegicus
Steventon, G.B.; Mitchell, S.C.
Phenylalanine 4-monooxygenase and the role of endobiotic metabolism enzymes in xenobiotic biotransformation
Expert. Opin. Drug Metab. Toxicol.
5
1213-1221
2009
Rattus norvegicus, Homo sapiens (P00439), Homo sapiens
Patel, N.; Iliadou, C.; Boonyapiwat, B.; Barlow, D.; Forbes, B.; Mitchell, S.; Steventon, G.
Enzyme kinetic and molecular modelling studies of sulphur-containing substrates of phenylalanine 4-monooxygenase
J. Enzyme Inhib. Med. Chem.
23
958-963
2008
Homo sapiens, Rattus norvegicus
Li, J.; Ilangovan, U.; Daubner, S.C.; Hinck, A.P.; Fitzpatrick, P.F.
Direct evidence for a phenylalanine site in the regulatory domain of phenylalanine hydroxylase
Arch. Biochem. Biophys.
505
250-255
2011
Rattus norvegicus
Li, J.; Dangott, L.J.; Fitzpatrick, P.F.
Regulation of phenylalanine hydroxylase: conformational changes upon phenylalanine binding detected by hydrogen/deuterium exchange and mass spectrometry
Biochemistry
49
3327-3335
2010
Rattus norvegicus (P04176)
Jaffe, E.K.; Stith, L.; Lawrence, S.H.; Andrake, M.; Dunbrack, R.L.
A new model for allosteric regulation of phenylalanine hydroxylase: implications for disease and therapeutics
Arch. Biochem. Biophys.
530
73-82
2013
Homo sapiens, Rattus norvegicus (P04176)
Roberts, K.M.; Pavon, J.A.; Fitzpatrick, P.F.
Kinetic mechanism of phenylalanine hydroxylase: intrinsic binding and rate constants from single-turnover experiments
Biochemistry
52
1062-1073
2013
Rattus norvegicus
Flydal, M.I.; Martinez, A.
Phenylalanine hydroxylase: function, structure, and regulation
IUBMB Life
65
341-349
2013
Caenorhabditis elegans, Legionella pneumophila, Homo sapiens (P00439), Homo sapiens, Rattus norvegicus (P04176), Chromobacterium violaceum (P30967), Colwellia psychrerythraea (Q47XN7), Legionella pneumophila 130b
Roberts, K.M.; Khan, C.A.; Hinck, C.S.; Fitzpatrick, P.F.
Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site
Biochemistry
53
7846-7853
2014
Rattus norvegicus
Zhang, S.; Hinck, A.P.; Fitzpatrick, P.F.
The amino acid specificity for activation of phenylalanine hydroxylase matches the specificity for stabilization of regulatory domain dimers
Biochemistry
54
5167-5174
2015
Rattus norvegicus (P04176)
Meisburger, S.P.; Taylor, A.B.; Khan, C.A.; Zhang, S.; Fitzpatrick, P.F.; Ando, N.
Domain movements upon activation of phenylalanine hydroxylase characterized by crystallography and chromatography-coupled small-angle X-ray scattering
J. Am. Chem. Soc.
138
6506-6516
2016
Homo sapiens (P00439), Rattus norvegicus (P04176)
Zhang, S.; Fitzpatrick, P.F.
Identification of the allosteric site for phenylalanine in rat phenylalanine hydroxylase
J. Biol. Chem.
291
7418-7425
2016
Rattus norvegicus (P04176)
Becher, I.; Werner, T.; Doce, C.; Zaal, E.A.; Toegel, I.; Khan, C.A.; Rueger, A.; Muelbaier, M.; Salzer, E.; Berkers, C.R.; Fitzpatrick, P.F.; Bantscheff, M.; Savitski, M.M.
Thermal profiling reveals phenylalanine hydroxylase as an off-target of panobinostat
Nat. Chem. Biol.
12
908-910
2016
Rattus norvegicus
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