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The isolation and properties of phenylalanine hydroxylase from rat liver.

ABSTRACT: Phenylalanine hydroxylase was prepared from rat liver and purified 200-fold to about 90% purity. All the enzymic activity of the liver appeared in a single protein of mol.wt. approx. 110000, but omission of dithiothreitol and of a preliminary filtration step to remove lipids resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. The K(m) and V(max.) values of the enzyme for phenylalanine, p-fluorophenylalanine and dimethyltetrahydropterin were measured; p-chlorophenylalanine inhibited the enzyme by competing with phenylalanine. Disc gel electrophoresis at pH7.2 showed a single protein band containing all the enzymic activity, but at pH8.7 the enzyme dissociated into two inactive fragments of similar but not identical molecular weight. The molecule of phenylalanine hydroxylase contained two atoms of iron, one atom of copper and one molecule of FAD; molybdenum was absent. Treatment with chelating agents showed that both non-haem iron and copper were necessary for enzymic activity. The molecule contained five thiol groups, and thiol-binding reagents inhibited the enzyme. Catalase or peroxidase enhanced enzymic activity fivefold; it is postulated that catalase (or other peroxidase) plays a part in the hydroxylation reaction independent of the protection by catalase of enzyme and cofactor from inactivation by a hydroperoxide.

SUBMITTER: Gillam SS

PROVIDER: S-EPMC1166337 | biostudies-other | 1974 Jun

REPOSITORIES: biostudies-other

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The isolation and properties of phenylalanine hydroxylase from human liver.

Project description:Phenylalanine hydroxylase was prepared from human foetal liver and purified 800-fold; it appeared to be essentially pure. The phenylalanine hydroxylase activity of the liver was confined to a single protein of mol.wt. approx. 108000, but omission of a preliminary filtration step resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. Human adult and full-term infant liver also contained a single phenylalanine hydroxylase with molecular weights and kinetic parameters the same as those of the foetal enzyme; foetal, newborn and adult phenylalanine hydroxylase are probably identical. The K(m) values for phenylalanine and cofactor were respectively one-quarter and twice those found for rat liver phenylalanine hydroxylase. As with the rat enzyme, human phenylalanine hydroxylase acted also on p-fluorophenylalanine, which was inhibitory at high concentrations, and p-chlorophenylalanine acted as an inhibitor competing with phenylalanine. Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding reagents inhibited the enzyme but, as with the rat enzyme, phenylalanine both stabilized the human enzyme and offered some protection against these inhibitors. It is hoped that isolation of the normal enzyme will further the study of phenylketonuria.

| S-EPMC1166338 | biostudies-other

Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase.

Project description:Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25-117) is the regulatory domain of PheH lacking residues 1-24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25-117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25-117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH.

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Isolation of phenylalanine hydroxylase-stimulating monoclonal antibody by rat-myeloma--rat-spleen-cell fusion.

Project description:A monoclonal antibody directed against monkey liver phenylalanine hydroxylase was produced by using a rat-myeloma--rat-spleen-cell-fusion system. This antibody showed the interesting property of increasing mammalian phenylalanine hydroxylase activity more than 2-fold. Perhaps monoclonal antibodies with this effect on other enzyme or proteins could be developed.

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Liver phenylalanine hydroxylase activity in relation to blood concentrations of tyrosine and phenylalanine in the rat.

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Allosteric regulation of phenylalanine hydroxylase.

Project description:The liver enzyme phenylalanine hydroxylase is responsible for conversion of excess phenylalanine in the diet to tyrosine. Phenylalanine hydroxylase is activated by phenylalanine; this activation is inhibited by the physiological reducing substrate tetrahydrobiopterin. Phosphorylation of Ser16 lowers the concentration of phenylalanine for activation. This review discusses the present understanding of the molecular details of the allosteric regulation of the enzyme.

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The effect of streptozotocin-induced diabetes on phenylalanine hydroxylase expression in rat liver.

Project description:The impact of experimentally induced diabetes on the expression of rat liver phenylalanine hydroxylase has been investigated. A significant elevation in maximal enzymic activity was observed in diabetes. This was associated with significant increases in the amount of enzyme, the phenylalanine hydroxylase-specific translational activity of hepatic RNA and the abundance of phenylalanine hydroxylase-specific mRNA. These changes in phenylalanine hydroxylase expression were not observed when diabetes was controlled by daily injections of insulin. These results are discussed in relation to the hormonal control of phenylalanine hydroxylase gene expression.

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The quantitative determination of phenylalanine hydroxylase in rat tissues. Its developmental formation in liver.

Project description:A sensitive method was developed for determining the phenylalanine hydroxylase activity of crude tissue preparations in the presence of optimum concentrations of the 6,7-dimethyl-5,6,7,8-tetrahydropterin cofactor (with ascorbate or dithiothreitol to maintain its reduced state) and substrate. Tissue distribution studies showed that, in addition to the liver, the kidney also contains significant phenylalanine hydroxylase activity, one-sixth (in rats) or half (in mice) as much per g as does the liver. The liver and the kidney enzyme have similar kinetic properties; both were located in the soluble phase and were inhibited by the nucleo-mitochondrial fraction. Phenylalanine hydroxylase, like most rat liver enzymes concerned with amino acid catabolism, develops late. On the 20th day of gestation, the liver (and the kidney) is devoid of phenylalanine hydroxylase and at birth contains 20% of the adult activity. During the second postnatal week of development, when the phenylalanine hydroxylase activity was about 40% of the adult value, an injection of cortisol doubled this value. Cortisol had no significant effect on phenylalanine hydroxylase in adult liver or on phenylalanine hydroxylase in kidney at any age.

| S-EPMC1178764 | biostudies-other

Phenylalanine hydroxylase misfolding and pharmacological chaperones.

Project description:Phenylketonuria (PKU) is a loss-of-function inborn error of metabolism. As many other inherited diseases the main pathologic mechanism in PKU is an enhanced tendency of the mutant phenylalanine hydroxylase (PAH) to misfold and undergo ubiquitin-dependent degradation. Recent alternative approaches with therapeutic potential for PKU aim at correcting the PAH misfolding, and in this respect pharmacological chaperones are the focus of increasing interest. These compounds, which often resemble the natural ligands and show mild competitive inhibition, can rescue the misfolded proteins by stimulating their renaturation in vivo. For PKU, a few studies have proven the stabilization of PKU-mutants in vitro, in cells, and in mice by pharmacological chaperones, which have been found either by using the tetrahydrobiopterin (BH(4)) cofactor as query structure for shape-focused virtual screening or by high-throughput screening of small compound libraries. Both approaches have revealed a number of compounds, most of which bind at the iron-binding site, competitively with respect to BH(4). Furthermore, PAH shares a number of ligands, such as BH(4), amino acid substrates and inhibitors, with the other aromatic amino acid hydroxylases: the neuronal/neuroendocrine enzymes tyrosine hydroxylase (TH) and the tryptophan hydroxylases (TPHs). Recent results indicate that the PAH-targeted pharmacological chaperones should also be tested on TH and the TPHs, and eventually be derivatized to avoid unwanted interactions with these other enzymes. After derivatization and validation in animal models, the PAH-chaperoning compounds represent novel possibilities in the treatment of PKU.

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Mouse phenylalanine hydroxylase. Homology and divergence from human phenylalanine hydroxylase.

Project description:The laboratory mouse represents an important model for the study of phenylalanine metabolism and the pathochemistry of phenylketonuria, yet mouse phenylalanine hydroxylase (PAH) has not been extensively studied. We report the cloning and sequencing of a mouse PAH cDNA, the expression of enzymic activity from the mouse PAH cDNA clone and the identification of mouse PAH and human PAH by two-dimensional PAGE of liver samples. These data confirm the expected homology of mouse PAH and human PAH and suggest differences in the primary sequence and the phosphorylation state of the two enzymes.

| S-EPMC1131302 | biostudies-other

The regulation of phenylalanine hydroxylase in rat tissues in vivo. Substrate- and cortisol-induced elevations in phenylalanine hydroxylase activity.

Project description:Injections of phenylalanine increased a 2.5-fold in 9 h the hepatic phenylalanine hydroxylase activity of 6-day-old or adult rats that had been pretreated (24h earlier) with p-chlorophenylalanine; without such pretreatment, phenylalanine did not raise the enzyme concentration. This difference is paralleled by the much greater extent to which the injected phenylalanine accumulated in livers of the pretreated compared with the normal animals. The hormonal induction of hepatic phenylalanine hydroxylase activity obeyed different rules: an injection of cortisol was without effect on adult livers but caused a threefold rise in phenylalanine hydroxylase activity of immature ones, both without and after pretreatment with p-chlorophenylalanine. In the latter instance, the effects of cortisol, and of phenylalanine were additive. Actinomycin inhibited the cortisol- but not the substrate-induced increase of phenylalanine hydroxylase, whereas puromycin inhibited both. The results indicate that substrate and hormone, two potential positive regulators of the amount of the hepatic (but not the renal) phenylalanine hydroxylase, act independently by two different mechanisms. The negative effector, p-chlorophenylalanine, also appears to interact with the synthetic (or degradative) machinery rather than with the existing phenylalanine hydroxylase molecules: 24h were required in vivo for an 85% decrease to ensue, and no inhibition occurred in vitro when incubating the enzyme with p-chlorophenylalanine or with liver extracts from p-chlorophenylalanine-treated rats.

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