Sandbox Reserved 768: Difference between revisions
From Proteopedia
Jump to navigationJump to search
Arwa Omary (talk | contribs) No edit summary |
Arwa Omary (talk | contribs) No edit summary |
||
| (12 intermediate revisions by the same user not shown) | |||
| Line 2: | Line 2: | ||
{{User:Michael_B._Goshe/Template_BCH455_555}} | {{User:Michael_B._Goshe/Template_BCH455_555}} | ||
<!-- PLEASE ADD YOUR CONTENT BELOW HERE --> | <!-- PLEASE ADD YOUR CONTENT BELOW HERE --> | ||
'''Phenylalanine Hydroxylase''' (also known as Phenylalanine-4-monooxygenase or simply PAH) is the enzyme that catalyzes the conversion of L-phenylalanine into L-tyrosine by hydroxylation (addition of an -OH group) of the aromatic side chain of phenyalanine. This reaction is the initial and rate-limiting step in the phenylalanine catabolism pathway. The tyrosine product (a non-essential amino acid) can then serve as a precursor to the synthesis of important neurotransmitters.<ref name= "flydal"> Flydal, Marte, and Aurora Martinez. "Phenylalanine Hydroxylase: Function, Structure, and Regulation." International Union of Biochemistry and Molecular Biology Journal 65.4 (2013): 341-349. Web. </ref>. PAH uses tetrahydrobiopterin (BH4)as a cofactor and has a nonheme iron atom bound to its active site. PAH is classified as an oxidoreductase, specifically enzyme class EC 1.14 since its mechanism of action involves the oxidation/reduction of its substrate. <ref name= "pdb"> http://www.rcsb.org/pdb/explore/explore.do?structureId=1J8U </ref>. | |||
== Phenylalanine Hydroxylase == | |||
'''Phenylalanine Hydroxylase''' (also known as Phenylalanine-4-monooxygenase or simply PAH) is the enzyme that catalyzes the conversion of L-phenylalanine into L-tyrosine by hydroxylation (addition of an -OH group) of the aromatic side chain of phenyalanine. This reaction is the initial and rate-limiting step in the phenylalanine catabolism pathway. | |||
<Structure load='2pah' size='500' frame='true' align='right' caption='Dimeric Unit of Phenylalanine Hydroxylase with the iron atom in the active sites in brown. ' scene='Insert optional scene name here'/> The tyrosine product (a non-essential amino acid) can then serve as a precursor to the synthesis of important neurotransmitters.<ref name= "flydal"> Flydal, Marte, and Aurora Martinez. "Phenylalanine Hydroxylase: Function, Structure, and Regulation." International Union of Biochemistry and Molecular Biology Journal 65.4 (2013): 341-349. Web. </ref>. | |||
PAH uses tetrahydrobiopterin (BH4) as a cofactor and has a nonheme iron atom bound to its active site. PAH is classified as an oxidoreductase, specifically enzyme class EC 1.14 since its mechanism of action involves the oxidation/reduction of its substrate. <ref name= "pdb"> http://www.rcsb.org/pdb/explore/explore.do?structureId=1J8U </ref>. | |||
PAH enzyme is present in the liver cells of humans and other mammals. It is also present in non-mammalian eukaryote organisms and some bacteria such as ''E.coli''. <ref name= "pdb"/>. Recently, it has been identified in some protozoans and slime molds, and even in nonflowering plants such as spinach from which it has been extracted and studied. <ref> Nair, P, and L Vining. "Phenylalanine Hydroxylase from Spinach Leaves." Phytochemistry 4.3 (1965): 401-411. Web. </ref>. | PAH enzyme is present in the liver cells of humans and other mammals. It is also present in non-mammalian eukaryote organisms and some bacteria such as ''E.coli''. <ref name= "pdb"/>. Recently, it has been identified in some protozoans and slime molds, and even in nonflowering plants such as spinach from which it has been extracted and studied. <ref> Nair, P, and L Vining. "Phenylalanine Hydroxylase from Spinach Leaves." Phytochemistry 4.3 (1965): 401-411. Web. </ref>. | ||
Mammalian PAH is a homo-tetrameric enzyme of 50 kDa subunits composed of two asymmetric dimeric units. The four subunits are connected to each other via a coiled-coil motif as shown in the diagram below. | Mammalian PAH is a homo-tetrameric enzyme of 50 kDa subunits composed of two asymmetric dimeric units. The four subunits are connected to each other via a coiled-coil motif as shown in the diagram below. | ||
[[Image:PAH tetramer complex.jpg]] | [[Image:PAH tetramer complex.jpg]] | ||
Each monomeric subunit is composed of three sites: the N-terminal, the catalytic site, and the C-terminal. <ref name= " flydal"/>. | Each monomeric subunit is composed of three sites: the N-terminal, the catalytic site, and the C-terminal. <ref name= " flydal"/>. | ||
| Line 15: | Line 24: | ||
== Phenylalanine Hydroxylase Mechanism of Action == | == Phenylalanine Hydroxylase Mechanism of Action == | ||
PAH, belonging to the oxidoreductase enzyme class, acts by oxidizing/reducing its substrate. Specifically, PAH acts on paired donors with pteridine being a donor and first incorporates one atom of molecular oxygen into the aromatic ring of phenylalanine. <ref name= "pdb"/>. Then, it reduces the second oxygen atom to water using the two electrons that are supplied by the BH4 cofactor. BH4 is also hydroxylated at each turnover to produce pterin-4a-carbinolamine (4a-OH-BH4), with consequent dissociation from the enzyme. 4a-OHBH4 is dehydrated and reduced back to BH4 by the action of the enzyme pterin carbinolamine dehydratase. <ref name= "flydal"/>. | PAH, belonging to the oxidoreductase enzyme class, acts by oxidizing/reducing its substrate. Specifically, PAH acts on paired donors with pteridine being a donor and first incorporates one atom of molecular oxygen into the aromatic ring of phenylalanine. <ref name= "pdb"/>. Then, it reduces the second oxygen atom to water using the two electrons that are supplied by the BH4 cofactor. BH4 is also hydroxylated at each turnover to produce pterin-4a-carbinolamine (4a-OH-BH4), with consequent dissociation from the enzyme. 4a-OHBH4 is dehydrated and reduced back to BH4 by the action of the enzyme pterin carbinolamine dehydratase. <ref name= "flydal"/>. | ||
| Line 28: | Line 38: | ||
== Phenylalanine Hydroxylase Structure == | == Phenylalanine Hydroxylase Structure == | ||
<Structure load='1j8u' size='500' frame='true' align='right' caption='Catalytic Domain of Human Phenylalanine Hydroxylase Fe(II) in Complex with Tetrahydrobiopterin' scene='Insert optional scene name here'/> | |||
Although the full-length structure of mammalian PAH has not yet been clearly known, a large part of it has been identified. <ref name= "flydal"/>. It was solved by means of crystallizing the protein (at pH=7) to perform X-ray diffraction using molecular replacement. The search model used was based on the crystal structure of tyrosine hydroxylase because of the similarity between the two enzymes. <ref name="fusetti"> Fusetti, Fabrizia, Heidi Erlandsen, Torgeir Flatmark, and Raymond Stevens. "Structure of Tetrameric Human Phenylalanine Hydroxylase and Its Implications for Phenylketonuria." The Journal of Biological Chemistry 273.27 (1998): 16962-16967. Web. </ref>. The R-factor recorded was 0.251 and the mean B (or temperature) value was 33.0. <ref name= "pdb"/>. | Although the full-length structure of mammalian PAH has not yet been clearly known, a large part of it has been identified. <ref name= "flydal"/>. It was solved by means of crystallizing the protein (at pH=7) to perform X-ray diffraction using molecular replacement. The search model used was based on the crystal structure of tyrosine hydroxylase because of the similarity between the two enzymes. <ref name="fusetti"> Fusetti, Fabrizia, Heidi Erlandsen, Torgeir Flatmark, and Raymond Stevens. "Structure of Tetrameric Human Phenylalanine Hydroxylase and Its Implications for Phenylketonuria." The Journal of Biological Chemistry 273.27 (1998): 16962-16967. Web. </ref>. The R-factor recorded was 0.251 and the mean B (or temperature) value was 33.0. <ref name= "pdb"/>. | ||
'''Structure Revealed''' | '''Structure Revealed''' | ||
| Line 41: | Line 53: | ||
The catalytic domain contains the active site of the enzyme. It is composed of 13 α-helices and 8 β-strands <ref name= "fusetti"/> and houses the binding sites for the nonheme iron atom, the cofactor, and substrate. The iron binds to two histidines (His285 and His290 in hPAH) and a glutamate (Glu330) in the deep cleft in the core of each monomer. <ref name= "flydal"/>. | The catalytic domain contains the active site of the enzyme. It is composed of 13 α-helices and 8 β-strands <ref name= "fusetti"/> and houses the binding sites for the nonheme iron atom, the cofactor, and substrate. The iron binds to two histidines (His285 and His290 in hPAH) and a glutamate (Glu330) in the deep cleft in the core of each monomer. <ref name= "flydal"/>. | ||
The C-terminal oligomerization or tetramerization domain begins with an antiparallel-sheet | The C-terminal oligomerization or tetramerization domain begins with an antiparallel-sheet | ||
(residues 411–414, 421–424) | (residues 411–414, 421–424) | ||
and is formed by a C-terminal “arm” consisting of two β-strands, forming a β-ribbon, and a 40 Å long α-helix. This C-terminal arm extends over an adjacent monomer, thus bringing the four helices (one from each monomer) into a closely packed anti-parallel coiled-coil motif in the center of the structure (as can be seen in the tetramer structure above). <ref name= "fusetti"/>. The assembly of the enzyme occurs through a swapping mechanism in which the secondary structural elements mutually switch their position to promote oligomerization. <ref name= "fusetti"/>. | and is formed by a C-terminal “arm” consisting of two β-strands, forming a β-ribbon, and a 40 Å long α-helix. This C-terminal arm extends over an adjacent monomer, thus bringing the four helices (one from each monomer) into a closely packed anti-parallel coiled-coil motif in the center of the structure (as can be seen in the tetramer structure above). <ref name= "fusetti"/>. The assembly of the enzyme occurs through a swapping mechanism in which the secondary structural elements mutually switch their position to promote oligomerization. <ref name= "fusetti"/>. | ||
The residues surrounding the iron atom in the active site are: PHE 254, ARG 270, HIS 285, GLU 286, TRP 326, and GLU 330. <ref name= "flydal"/>. The illustration below highlights these residues and their relative positions to the iron atom, BH4 cofactor, and the L-Phenyalanine substrate. | |||
[[Image:Active residues.jpg]] | |||
== PAH Mutations == | |||
Mutations and changes in the β-ribbon region have major detrimental effects on the enzyme stability. The mutations that lead | |||
to Phenylketonuria are most likely due to mutations in the PAH gene that cause changes at the junction between the catalytic and tetramerization domains of the enzyme. <ref name= "fusetti"/>. This leads to loss of enzyme stability and extensive misfolding which subsequently results in enzyme malfunction. <ref name= "flydal"/>. The monomeric image below highlights the regions in the active site that, if mutated, would destroy enzyme activity and lead to phenylketonuria. The yellow atom is iron, the green structure is the BH4 cofactor, | |||
and the red sites are a few examples of mutation regions. | |||
[[Image:mutation sites.jpg]] | |||
== Phenylketonuria == | |||
'''Overview''' | |||
Phenylketonuria (PKU) is the most common inborn error of amino acid metabolism. <ref name="lendly"> Lendley, Fred, Anthony DeLilla, and Savio Woo. "Molecular Biology of Phenylalanine Hydroxylase and Phenylketonuria." Trends in Genetics 1(1985): 309-313. Web.</ref>. It is genetic autosomal recessive disorder and a relatively common condition affecting approximately 1 in 10,000 newborn infants in the USA and Europe. <ref name="lendly"/>. The Phenylhydroxylase enzyme gene consists of 13 exons and introns and when both alleles are mutated (resulting in the mutated regions highlighted in the enzyme active site illustration above), phenylketonuria arises.<ref name="blau"> Blau, Nenad, Francjan Van Spronsen, and Harvey Levy. "Phenylkentonuria." The Lancet 376.9750 (2010): 1417-1427. Web. </ref>. The two mutations may occur in any of the exons, intervening introns, or perhaps in other regions such as the promoter. | |||
Loss of PAH activity results in increased concentrations of phenylalanine in the blood and toxics in the brain. In addition, dysfunctional PAH leads to the appearance, in urine, of metabolites that arise from the transamination of L-Phe to phenylpyruvate. <ref name= "flydal"/>. Phenylketonuria is classified by the severity of hyperphenylalaninaemia.<ref name= "blau"/>. The normal range of blood phenylalanine concentrations is 50–110 μmol/L. Individuals with blood phenylalanine concentrations of 120–600 μmol/L are classified as having mild hyperphenylalaninaemia; those with concentrations of 600–1200 μmol/L are classified as mild phenylketonuria; and concentrations above 1200 μmol/L denote classic phenylketonuria. <ref name= "blau"/>. | |||
'''Symptoms''' | |||
The common signs and symptoms associated with phenylketonuria include: progressive intellectual impairment, depression, and purposeless movement. <ref name= "flydal"/>. Other symptoms that have been noted encompass eczematous rash, autism, seizures, and motor deficits. <ref name= "blau"/>. However, the classic symptoms are those of neurophysiological and psychiatric impairments. | |||
'''Molecular Explanation''' | |||
The correlation between PAH malfunction and neurological deterioration can be explained as follows. The accumulation of phenylalanine inhibits the function of the aminoacid transporter 1 (LAT 1) at the brain's entry. <ref name= "blau"/>. This is detrimental because such transporter mediates the entry of other amino acids such as tyrosine and tryptophan into the brain. <ref name= "blau"/>. Once this transporter carrier is impaired, such amino acids are unable to cross into the brain. These aromatic amino acids serve as precursors to the synthesis of important neurotransmittors- tyrosine is a precursor for dopamine and norephinephrine, and tryptophan is a precursor for serotonin. Their absence then immensely disrupts the production of important neurotransmitters, hence resulting in the neurological impairment seen with phenylketonuria. | |||
'''Treatment''' | |||
There does not seem to be any medicinal treatment available to alleviate the symptoms of phenylketonuria. The most important step in managing this condition is dietary phenylalanine monitoring and restriction and this usually begins immediately after confirmation of hyperphenylalaninaemia in a neonate. <ref name= "blau"/>. Patients with phenylketonuria have to accept the phenylalanine-free formula and avoid foods rich in protein as well as foods and drinks containing aspartame, flour, and soya. Low-protein natural foods such as potatoes, some vegetables, and most cereals can be eaten but only in severely restricted amounts. Luckily, low-protein variants of some foods exist, such as low-protein bread and low-protein pasta. <ref name= "blau"/>. Diet monitoring and management has been shown to very successful in controlling phenylketonuria and halting its progression. | |||
== References == | |||
<references /> | |||
