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Structure
Primary Structure:
The primary structure of each monomer of phenylalanine hydroxylase contains 452 residues, weighing around 52kDa.
(Include image of entire amino acid sequence)
Secondary Structure:
PAH contains right-handed alpha helices and antiparallel beta-strands in its secondary structure. There are some amino acids that don't have any secondary structure, and these are found in the loop containing regions. The loop containing regions are residues L42-V45, D59-H69, S70-D75, and H82-V90.
(include image of secondary structure)
Tertiary Structure:
The tertiary structure of each monomer of PAH is organized from 2 alpha helices and 4 beta-strands into an alpha-beta sandwich motif (BaBBaB fold). The structural motif of an alpha-beta sandwich motif has the 4 antiparallel beta-strands flanked on one side by the 2 alpha-helices. The tertiary structure of a phenylalanine hydroxylase protein is built from an N-terminal regulatory domain (residues 1-117), a catalytic domain (residues 118-410), and a tetramerization domain (residues 411-452). The catalytic domain includes the binding sites for iron, substrate and cofactor.The binding sites are at residues 285, 290, and 330. (explain what the tetramerization domain is) The ACT domain is in the N-terminal regulatory domain where proposed enzyme binding to an allosteric site (residues 3-11).
Quaternary Structure:
The quaternary structure of PAH is a homotetramer, dimer of dimers. It is a multidomain, homo-oligomeric protein with dihedral (D2) symmetry.
The substrate of phenylalanine hydroxylase is the amino acid L-phenylalanine. Phenylalanine binds between the regulatory domain and the interacting catalytic domain, near the sequence binding motif. The activation of PAH by L-phenylalanine induces a large conformational change, but a slow global conformational change (is it large in magnitude but slow overall?) Full activation of PAH involves the shift and dimerization of the regulatory domains.
PAH is an iron (Fe3+) containing enzyme. The iron binds to 2 histidines at the active site. The cofactors of PAH include 6R-L-erythro-tetrahydrobiopterin (BH4) and oxygen. BH4 is sandwiched between hydrophobic residues and forms several hydrogen bonds with the N-terminal autoregulatory tail. BH4 binding causes a limited conformational change (mostly constrained to the N-terminal tail). PAH lacking this tail is not regulated by either BH4 or L-phenylalanine and is constitutively active. The BH4 binding-site is flanked by the N-terminal (residues 21-32), the active-site lid (130-150), the Fe+2-coordinating residues, the Beta 6-alpha 7 loop (residues 245-251), and F254.
Tetrahydrobiopterin induces a negative heterotropic allosteric effect on the enzyme, which is observed as the activation rate is slower for the BH4 holoprotein than compared to the unbound enzyme. Prior to BH4 binding, (PAH unbound state) a polar and salt-bridge interaction network links the three PAH domains.
== Function == The genetic information that codes for the production of phenylalanine hydroxylase is found on chromosome 12 and contains 13 exons. PAH is a metabolic enzyme contained in liver cells that catalyzes the hydroxylation reaction of the amino acid L-Phenylalanine to L-Tyrosine. This protein specifically catalyzes the rate-limiting step in the phenylalanine catabolism, which is the para-hydroxylation step of the aromatic side chain. This catalysis is done by hydroxylation of its substrate by incorporation of one oxygen atom into the aromatic ring, and the final reaction includes the reduction of the second oxygen atom to water using electrons supplied by tetrahydrobiopterin (BH4). BH4 functions as a co-substrate that is hydroxylated at each turnover to pterin-4a-carbinolamine (4a-OH-BH4), with consequent dissociation from the enzyme.
The major regulatory mechanisms of phenylalanine hydroxylase include activation of phenylalanine inhibition by BH4, and additional activation by phosphorylation. Phosphorylation acts as a mediator of phenylalanine activation by decreasing phenylalanine concentration required to activate enzyme phosphorylation at Ser16. Substrate activation and positive homotropic allosterics for phenylalanine binding involves all three functional domains and all four subunits in the holoenzyme. The hypothesized cause of the phenylalanine activation mechanism is that homotropic binding of phenylalanine at the active site and the regulatory domain is involved in cooperativity through the interactions with the catalytic and oligomerization domains. Phenylalanine binds to an allosteric site, besides the active site, on the regulatory domain, which induces large conformational changes. The allosteric regulation is necessary to maintain phenylalanine levels below neurotoxic levels. BH4 acts as a negative allosteric regulator by blocking phenylalanine activation, however, BH4 binding to a Phe-activated form of PAH results in positive cooperativity.
== Phenylketonuria == L-Tyrosine is the precursor to neurotransmitters such as epinephrine, dopamine, and serotonin. It is essential that L-phenylalanine is converted into L-tyrosine by the hydroxylation reaction. In order for this conversion to be successful, the enzyme phenylalanine hydroxylase needs to be able to function properly. PAH depletion or mutation leads to excessive accumulation of toxic L-Phe levels. However, normal physiological plasmatic levels of L-phenylalanine are less than 120 micromolar. When the enzyme PAH doesn’t function currently, the autosomal recessive metabolic disorder Phenylketonuria (PKU) occurs. PKU is a congenital disorder characterized by excessive amounts of L-phenylalanine that buildup to neurotoxic amounts leading to cognitive disability and neurological impairment, including profound mental retardation, seizures, microcephaly, and delayed development. The severity of PKU is dependent upon the severity of the enzyme’s mutation. PAH mutations result in reduced enzyme activity and stability and some alter its oligomeric state. These mutations spread throughout the 3D structure, but most are located in the catalytic domain. Loss of enzymatic function is caused mainly by folding defects that lead to decreased protein stability.
Treatments for phenylketonuria include a lifelong diet avoiding foods containing phenylalanine and supplementation of synthetic formations of the cofactor tetrahydrobiopterin (BH4).
Relevance
Structural highlights
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