Phenylalanine-4-hydroxylase
Iris Pardue '24 and Safia Mohan '24
Contents:
I. Introduction
The gene PAH encodes the enzyme phenylalanine-4-hydroxylase, a
protein responsible for the conversion of phenylalanine to tyrosine.
It plays an important role in the metabolism of amino acids throughout
the body. It is a member of the family of amino acid hydroxylases, and
shares several structural and functional motifs.
Mutations in this enzyme result in phenylketonuria or PKU, an
autosomal recessive genetic condition that reduces the ability of
the affected individual to metabolize phenylalanine. The disease can
either manifest in its classical form where the activity of the
enzyme itself is affected; or in variant PKU, where enzyme function
is not directly impeded but the a change in the presence of its
cofactors reduces its efficiency. The classical form is incurable
but can be treated with low-phenylalanine diets, while the variant
form can sometimes be treated with supplements of the necessary
cofactors.
II. General Structure
Phenylalanine-4-hydroxylase is a 77.4 kDa tetramer
composed of two identical dimers
. Each monomer has its own tetrameric
and catalytic domains.
The tetrameric domain is where all 4 monomers interact to form the
tetramer. The catalytic domain is composed of the rest of the
compund, it is the reigon that binds Fe
and interacts with phyenylalanine.
III. Catalyst Binding
The Fe ion binds HIS285, HIS290,
and GLU330 in order to properly coordinate it to the correct
position.
It rests in close proximity to BH4
(tetrahydrobiopterin), a molecule that acts as a necessary
catalyst for hydroxylation along with enzyme stabilization, but
interestingly slows enzyme folding into the active conformation. BH4
is stabilized by H-bond interactions with S23, G247, L249, S251, and
Y377; along with pi-stacking interactions with F254.
Notably,E286 is involved in bonding with both catalytic molecules
through interactions with water.
IV. Metabolite Binding
Phenylalanine-4-hydroxylase is catalyzed by
Fe(II) ion and BH4. The
hydroxylation is thought to proceed via a Fe(IV)=O intermediate
formed from the Fe(II) ion and free
O2, oxidizing the BH4 in the process.
This Fe(IV) species then goes on to
attack the phenyl ring of the phenylalanine, disrupting its
aromaticity to form an Fe-O-phenyl bridge. This decomposes and
releases the Fe ion to form an
O=phenyl bond, which tautomerizes to HO-phenyl to restore the
aromaticity and form the end product of tyrosine.
V. Mutated Regions
Mutations in the enzyme are well studied and important to
characterizing the pathology of PKU, and many revolve around binding
of the catalytic molecules described above. For example, the
mutation of F254 to alanine
resulted in a significant decrease in the affinity of the enzyme for
BH4, and mutation of the same position
to leucine extremely hindered the ability of the enzyme to bind
substrate phenylalanine. Mutations in E286
to either alanine or glutamine
both significantly decreased reaction rate as well, likely due to
its importance in coordinating binding of both catalysts.
Understanding these mutations and interactions allows us to better
treat PKU cases by understanding how and when supplementation with
synthetic analogs of BH4 can restore
enzyme function, and when that treatment may be ineffective.
VI. References
Fabrizia
Fusetti, Heidi Erlandsen, Torgeir Flatmark, and
Raymond C. Stevens. 1998. Structure of Tetrameric
Human Phenylalanine Hydroxylase and Its Implications
for Phenylketonuria. The
Journal of Biological Chemistry
273: 16962-16967
Marte Innselset Flydal, Martin
Alcoro-Pages, Fredrik Gullaksen Johannessen, Siseth
Martinez-Caballerp, Lars Skjaerven, Rafael Fernandez-Leiro,
Aurora Martinez, and Juan A. Hermoso. 2019. Structure of
full-length human phenylalanine hydroxylase in complex with
tetrahydrobiopterin. Proceedings of the National Academy of
Sciences 116: 11229-111234.
Heidi Erlandsen, Elisa Bjorgo, Torgeir
Flatmark, and Raymond C. Stevens. 2000. Crystal
Structure and Site-Specific Mutagenesis of Pterin-Bound Human
Phenylalanine Hydroxylase. Biochemistry 39: 2208-2217.
Fitzpatrick, P. F. (2003). Mechanism of Aromatic Amino Acid
Hydroxylation. Biochemistry, 42(48), 14083–14091.
https://doi.org/10.1021/bi035656u
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