Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phenylketonuria (PKU) is a metabolic disorder that results from a deficiency of hepatic phenylalanine hydroxylase (PAH). Identification of the PKU genotype is useful for predicting clinical PKU phenotype. More than 400 mutations resulting in PAH deficiency have been reported worldwide. We used a genedetecting instrument to identify the nine prevalent Japanese mutations in the PAH gene among 31 PKU patients as a preliminary study. This instrument can automatically detect mutations through the use of allelespecific oligonucleotide (ASO) capture probes, and gave results comparable to those of sequencing studies. Each country has uniquely prevalent and specific mutations causing PKU, and less than 50 types of such mutations are generally present in each country. Early genotyping of PKU makes it possible to identify the phenotype and select the optimal therapy for the disease. For early genotyping, the instrumental method described here shortens the time required for genotyping based on mRNA and/or genomic DNA of PKU parents.
Mol Biotechnol 2003 Jul
PMID:Rapid single-base mismatch detection in genotyping for phenylketonuria. 1277 91

The crystal structures of the catalytic domain of human phenylalanine hydroxylase (hPheOH) in complex with the physiological cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and the substrate analogues 3-(2-thienyl)-L-alanine (THA) or L-norleucine (NLE) have been determined at 2.0A resolution. The ternary THA complex confirms a previous 2.5A structure, and the ternary NLE complex shows that similar large conformational changes occur on binding of NLE as those observed for THA. Both structures demonstrate that substrate binding triggers structural changes throughout the entire protomer, including the displacement of Tyr138 from a surface position to a buried position at the active site, with a maximum displacement of 20.7A for its hydroxyl group. Two hinge-bending regions, centred at Leu197 and Asn223, act in consort upon substrate binding to create further large structural changes for parts of the C terminus. Thus, THA/L-Phe binding to the active site is likely to represent the epicentre of the global conformational changes observed in the full-length tetrameric enzyme. The carboxyl and amino groups of THA and NLE are positioned identically in the two structures, supporting the conclusion that these groups are of key importance in substrate binding, thus explaining the broad non-physiological substrate specificity observed for artificially activated forms of the enzyme. However, the specific activity with NLE as the substrate was only about 5% of that with THA, which is explained by the different affinities of binding and different catalytic turnover.
J Mol Biol 2003 Oct 31
PMID:2.0A resolution crystal structures of the ternary complexes of human phenylalanine hydroxylase catalytic domain with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine or L-norleucine: substrate specificity and molecular motions related to substrate binding. 1456 34

Tetrahydrobiopterin (BH(4)) is widely used as a therapeutic agent in patients with BH(4) deficiencies and mild forms of phenylketonuria (PKU) and there is an increasing need for the measurement of its plasma concentrations in patients with cardiovascular disorders. We measured BH(4) and total biopterin in dithioerythritol (DTE) pretreated plasma from four adults after oral administration of BH(4) (2, 10, and 20mg/kg body weight) using the differential iodine oxidation method. About 80% (range 64.8-92.2% ) of total biopterin was found as BH(4) when analyzed immediately after blood sampling. Compared with ascorbic acid as an antioxidant, DTE was more protective against oxidation of BH(4), particularly in samples stored over a period of 8 months. Without antioxidant (DTE or ascorbic acid) almost no BH(4) was detected. Furthermore, BH(4) and total biopterin were measured at different time intervals (up to 33 h after oral administration) and pharmacokinetic parameters T(max) (1-4h), C(max) (258.7-259.0 nmol/L biopterin at a dosage of 10mg/kg), and area under the curve (AUC=1708-1958 nmol(*)h/L up to T=10h) were estimated. The elimination half-life time was calculated to be 3.3-5.1h. Doubling the BH(4) dosage to 20mg/kg resulted in 60% higher AUC while sublingual BH(4) application (2mg/kg) resulted in 58-76% higher BH(4) plasma concentrations when compared with oral administration. These preliminary data suggest that in patients with BH(4) cofactor defects and BH(4)-responsive phenylalanine hydroxylase deficiency, BH(4) should be given in at least two to three daily doses and that sublingual administration may lower the required BH(4) dosage and subsequently the cost of treatment. Due to inter individual differences in pharmacokinetic properties, in some patients with hyperphenylalaninemia and mild PKU plasma BH(4) levels may be not high enough to fully activate the liver phenylalanine hydroxylase and thus lower blood phenylalanine levels. Assessment of plasma BH(4) or total biopterin concentrations may be a good way to control the efficacy of the loading test.
Mol Genet Metab 2004 Jan
PMID:Plasma tetrahydrobiopterin and its pharmacokinetic following oral administration. 1472 90

Tetrahydrobiopterin (BH(4)) is a required cofactor for the enzymatic activity of phenylalanine hydroxylase (PAH) and is synthesized de novo from GTP in several tissues. Heterologous expression of PAH in tissues other than liver is a potential novel therapy for human phenylketonuria that is completely dependent upon BH(4) supply in the PAH-expressing tissue. Previous experiments with liver PAH-deficient transgenic mice that expressed PAH in skeletal muscle demonstrated transient correction of hyperphenylalaninemia only with hourly parenteral BH(4) administration. In this report, the fate of intravenously administered BH(4) is examined. The conclusions are that (1) BH(4) administered intravenously is rapidly taken up by liver and kidney, and (2) uptake of BH(4) into muscle is relatively low. The levels of BH(4) achieved in skeletal muscle following IV injection are only 10% of the amount expected were BH(4) freely and equally distributed across all tissues. The half-life of BH(4) in muscle is approximately 30 min, necessitating repeated injections to maintain muscle BH(4) content sufficient to support phenylalanine hydroxylation. The efficacy of heterologous muscle-directed gene therapy for the treatment of PKU will likely be limited by the BH(4) supply in PAH-expressing muscle.
Mol Genet Metab 2004 Jan
PMID:The fate of intravenously administered tetrahydrobiopterin and its implications for heterologous gene therapy of phenylketonuria. 1472 91

We report on the long-term follow-up of the first Italian patient with the tetrahydrobiopterin (BH4)-responsive type of phenylalanine hydroxylase deficiency (R243X/Y414C genotype). The patient was diagnosed by the newborn screening for phenylketonuria (PKU) and with a positive BH4 loading test. Introduction of BH4 (initially 10 and later 20 mg/kg/day) in addition to reduced low-phenylalanine diet resulted in therapeutic plasma phenylalanine concentrations (<340 micromol/L). Very good compliance and no side effects in this patient demonstrate the great potential of BH4 in the treatment of some patients with mild PKU.
Mol Genet Metab 2004 Feb
PMID:Long-term follow-up of a patient with mild tetrahydrobiopterin-responsive phenylketonuria. 1474 Nov 96

Phenylketonuria (PKU) is a disease in which phenylalanine and phenylalanine-derived metabolites build up to neurotoxic levels due to mutations in the phenylalanine hydroxylase gene (PAH). Enzyme replacement therapy is a viable option to supply active PAH. However, the inherent protease sensitivity and potential immunogenicity of PAH have precluded adoption of this approach. In this report, we have used polyethylene glycol derivatization (PEGylation) to produce protected forms of PAH for potential therapeutic use. Three recombinantly produced PAH enzymes were reacted with activated PEG species, with the aim of developing a stable and active PKU enzyme replacement. Tetrameric full-length human PAH, dimeric double-truncated (DeltaN102-DeltaC428) human PAH, and monomeric Chromobacterium violaceum PAH were PEGylated with succinimidyl succinate polyethylene glycol of molecular weight 5000 or 20,000 Da. Characterization of the PEGylated species was accomplished with MALDI-TOF mass spectrometry, SDS-PAGE, and specific activity measurements using ESI mass spectrometry. All PEG-derivatized PAH species retained catalytic activity, and, at low numbers of PEG molecules attached, these PEGylated PAH proteins were found to be more active and more stable than their non-derivatized PAH counterparts.
Mol Ther 2004 Jan
PMID:Toward PKU enzyme replacement therapy: PEGylation with activity retention for three forms of recombinant phenylalanine hydroxylase. 1474 85

Phenylketonuria (PKU) is an inborn error of amino acid metabolism caused by phenylalanine hydroxylase (PAH) deficiency. Dietary treatment has been the cornerstone for controlling systemic phenylalanine (Phe) levels in PKU for the past 4 decades. Over the years, it has become clear that blood Phe concentration needs to be controlled for the life of the patient, a difficult task taking into consideration that the diet becomes very difficult to maintain. Therefore alternative models of therapy are being pursued. This review describes the progress made in enzyme replacement therapy for PKU. Two modalities are discussed, PAH and phenylalanine ammonia-lyase PAH. Developing stable and functional forms of both enzymes has proven difficult, but recent success in producing polyethylene glycol-modified forms of active and stable PAH shows promise.
Mol Ther 2004 Aug
PMID:Trends in enzyme therapy for phenylketonuria. 1529 68

We previously proposed a novel disease entity, tetrahydrobiopterin (BH4)-responsive phenylalanine hydroxylase (PAH) deficiency, in which administration of BH4 reduced elevated levels of serum phenylalanine [J. Pediatr. 135 (1999) 375-378]. Subsequent reports indicate that the prevalence of BH4-responsive PAH deficiency is much higher than initially anticipated. Although growing attention surrounds treatment with BH4, little is known about the mechanism of BH4 responsiveness. An early report indicates that BH4 concentration in rat liver was 5 microM where Km for BH4 of rat PAH was estimated to be 25 microM in an oxidation experiment using a liver slice, suggesting relative insufficiency of BH4 in liver in vivo. In the present study, we developed a breath test for mice using [1-13C]phenylalanine in order to examine the BH4 responsiveness of normal PAH in vivo. The reliability of the test was verified using BTBR mice and its mutant strain lacking PAH activity, Pahenu2. BH4 supplementation significantly enhanced 13CO2 production in C57BL/6 mice when phenylalanine was pre-loaded. Furthermore, BH4 apparently activated PAH in just 5 min. These observations suggest that submaximal PAH activity occurs at the physiological concentrations of BH4 in vivo, and that PAH activity can be rapidly enhanced by supplementation with BH4. Thus, we propose a possible hypothesis that the responsiveness to BH4 in patients with PAH deficiency is due to the fact that suboptimal physiological concentrations of BH4 are normally present in hepatocytes and the enhancement of the residual activity may be associated with a wide range of mutations.
Mol Genet Metab
PMID:Wild-type phenylalanine hydroxylase activity is enhanced by tetrahydrobiopterin supplementation in vivo: an implication for therapeutic basis of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency. 1546 29

Tetrahydrobiopterin (BH4) responsiveness in patients with mutations in the phenylalanine hydroxylase (PAH) gene is a recently recognized subtype of hyperphenylalaninemia characterized by a positive BH4 loading test. According to recent estimates, this phenotype may be quite common, suggesting that a large group of individuals may benefit from BH4 substitution, eliminating the need of life-long dietary restrictions. This underscores the importance of identifying BH4-responsive patients in each population, establishing the association with specific PAH mutations. In this work, we describe the results of a pilot study performed with 31 Spanish PAH-deficient patients subjected to a BH4 loading test. Overall, 11/31 (37%) showed a positive response with a 30% decrease in blood Phe levels 8 h after the BH4 challenge, and three additional patients, considered slow responders, showed this decrease only after 12-16 h. We report for the first time a patient homozygous for a splicing mutation with a slow response, suggesting an effect of BH4 supplementation on PAH gene expression. Most of the responsive patients belong to the mild hyperphenylalaninemia (MHP) or mild phenylketonuria phenotypic groups. In MHP patients we report for the first time the results of parallel single Phe doses confirming the utility of these analyses for a better evaluation of the response. Genotype analysis confirms the involvement in the response of specific mutations (D415N, S87R, R176L, E390G, and A309V) present in hemizygous patients, and provide relevant information for the discussion of the potential mechanisms underlying BH4 responsiveness.
Mol Genet Metab
PMID:Tetrahydrobiopterin responsiveness: results of the BH4 loading test in 31 Spanish PKU patients and correlation with their genotype. 1546 30

Melanin formation has a significant influence on mosquito vector competence by limiting the development of metazoan parasites. Tyrosine, the rate-limiting substrate of melanin production, can be obtained exogenously or derived from phenylalanine by phenylalanine hydroxylase (PAH). The characteristics of this defense mechanism, such as temporal expression of constituent enzymes involved in the biosynthetic pathway, can vary considerably between mosquito species. We investigated the functional role of PAH in the melanotic encapsulation response in Aedes aegypti and Armigeres subalbatus, two mosquito species with markedly different melanization responses. We used double-stranded RNA (dsRNA) to knock down PAH and observed the phenotypic effects on melanin formation. PAH transcripts were dramatically reduced in both mosquito species after gene knock down. The abundance of PAH proteins was decreased in gene knockdown mosquitoes that were inoculated with Dirofilaria immitis microfilariae (mf) as compared to inoculation controls. A significant reduction of mf melanization also was observed in these knockdown mosquitoes as compared to inoculation controls. Our data suggest that PAH is required for a fully functional melanotic encapsulation response in both mosquito vectors.
Insect Biochem Mol Biol 2004 Dec
PMID:The role of phenylalanine hydroxylase in melanotic encapsulation of filarial worms in two species of mosquitoes. 1554 46


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