Download Phenylketonuria (PKU)

Phenylketonuria (PKU)
Sanketh Proddutur
What is PKU?
•
Phenylketonuria (PKU) is an
inherited error of metabolism
caused by a deficiency in the
enzyme phenylalanine
hydroxylase (PAH).
•
phenylalanine hydroxylase
converts the amino acid
phenylalanine to tyrosine, another
amino acid.
Pathology
• mutations in the human gene (chromosome
12.q22-24.2), encoding PAH result in the
autosomal recessively inherited disease
hyperphenylalaninemia (HPA)
• The resulting phenotypes can range in severity
from mild hyperphenylalaninemia (HPA)
• classic PKU which inevitably leads to mental
retardation if left untreated.
Symptoms
Newborn infants develop signs of PKU within a few months
of birth if untreated:
• Mental retardation
• Behavioral or social problems
• Seizures, tremors or jerking movements in the arms and
legs
• Rocking
• Hyperactivity
• Stunted growth
Symptoms (contnd)
•
•
•
•
•
Skin rashes (eczema)
Small head size (microcephaly)
Vomiting
A musty odor in the child's breath, skin or urine
Fair skin and blue eyes
Etiology
Human phenylalanine hydroxylase converts
the essential amino acid L-phenylalanine
(L-Phe) into L-tyrosine using the co-factor
(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin
(BH4) and molecular oxygen.
Any disruption in this mechanism can lead to
Hyperphenylalaninemia.
Etiology (contnd)
99% of the mutations that disrupt this
mechanism can be traced back to the PAH
gene, 1% of the mutations are in the
genes encoding the enzymes which are
involved in the regeneration of the cofactor
BH4, which is a vital co-substrate of PAH.
BH4 regeneration
BH4 is a cofactor of PAH and its regeneration is
vital for the ongoing reaction.
PAH
Liver PAH is reported to exist in
solution as a pH dependent
equilibrium between homo-dimers
and the active homo-tetramers.
Four identical molecules of
phenylalanine hydroxylase interact
to form the tetramer, which is the
functional unit for this enzyme.
PAH (contnd)
Each monomer polypeptide is composed of
three domains
• An N-terminal regulatory domain
(residues 1-142)
• A central catalytic domain (residues 143410) which includes the active site iron
• A C-terminal tetramerization domain
(residues 411-452).
The regulatory domain consists
of a four stranded anti-parallel
β-sheet flanked on side by two
short α-helices and on the other
side by the catalytic domain
The catalytic domain houses the
active site iron in a novel basket like
arrangement of 14 α-helices and 8-β
strands
the short tetramerization domain is formed
by a C-terminal "arm" of two -strands
forming a -ribbon, and a long -helix and is
responsible for assembling the four chains
into the tetramer.
Catalytic domain
The active site iron is ligated in an octahedral manner and bound to His285, His290,
and 1 oxygen atom in Glu330 as well as three water molecules
Fe(II) ion
His 290
Glu 330
His 285
Catalytic domain (contnd)
A tunnel
adjacent to
the active
site might be
responsible
for directing
the
substrate.
Catalytic mechanism
In order to hydroxylate the unactivated Phenylalanine to Tyrosine, PAH
incorporates one atom of oxygen from molecular oxygen into the
substrate and reduces the other atom to water.
In the process, the BH4 cofactor undergoes a two-electron oxidation
to form quinonoid dihydropterin (q-BH2) which is then regenerated
back to BH4.
How exactly does this take place?
We don’t know!
It is however speculated that the binding of the
substrate is cooperative, that is the tetramer
undergoes synergistic conformational changes to
bind the substrates subsequently hydroxylating the
Phe residue.
Binding of the cofactor
The crystal structure of the dimeric catalytic domain (residues 118-424)
of human PAH cocrystallized with the oxidized form of the cofactor
(7,8-dihydro-L-biopterin, BH2), has been determined at 2.0 Å
resolution.
The pterin binds close to the catalytic iron and forms an extensive
H-bond network with Ala322, Gly247, Leu249 and Glu286.
The phenyl ring of Tyr325 establishes hydrophobic contacts with the
pterin, and thus contributes to the correct positioning of the pterin
cofactor for catalysis, furthermore this also ensures that the pterin
ring forms an aromatic pi-stacking interaction with Phe254.
Tests
• Guthrie card bloodspot: is the standard test for PKU. Blood taken
from the infant’s heel after first breast feeding is assayed for high
levels of Phe.
• tandem mass spectrometry (often abbreviated as MS/MS). This
technology can detect the blood components that are elevated in
certain disorders, and is capable of screening for more than 20
inherited metabolic disorders with a single test.
• Amniocentesis: is a form of prenatal diagnosis in which DNA
extracted from fetal cells obtained from the amniotic fluid at about
15-18 weeks' gestation period.
Treatment
• Restriction of dietary phenylalanine: is the most obvious treatment for
this disorder and also the most prevalent. Phe is ubiquitous across a variety
of food groups but is especially prevalent in proteins. A typical Pherestricted diet would be rich in fruits and some amounts of potato and milk
to maintain a phe concentration of about 2-6 mg/dL supplemented by a
.
phe-free protein supplement All kinds of meat, cheese and flour
products are forbidden.
Prognosis: is fairly good if the diet is strictly followed into adulthood.
Patients who discontinued after late adolescence.
• Other potential treatments being investigated are gene therapy, BH4
and phenylalanine lyase administration.
References
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Erlandsen, H. and Stevens, R.C. (1999) The structural basis of phenylketonuria.
Molecular Genetics and Metabolism 68, 103-25.
F.Fusetti, H.Erlandsen, T.Flatmark, R.C.Stevens. (1998) Structure of Tetrameric
Human Phenylalanine Hydroxylase and its Implications for Phenylketonuria.
J.Biol.Chem. 273, 16962
O.A.Andersen, T.Flatmark, E.Hough. (2001) High Resolution Crystal Structures of the
Catalytic Domain of Human Phenylalanine Hydroxylase in its Catalytically Active
Fe(II) Form and Binary Complex with Tetrahydrobiopterin. J.Mol.Biol. 314; 266
H.Erlandsen, E.Bjorgo, T.Flatmark, R.C.Stevens(2000). Crystal Structure and SiteSpecific Mutagenesis of Pterin-Bound Human Phenylalanine Hydroxylase.
Biochemistry 2208;