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Phenylalanine hydroxylase in liver cells. Correlation of glucagon-stimulated enzyme phosphorylation with expressed activity.

ABSTRACT: Phenylalanine is transported rapidly into, but is not concentrated by, liver cells. Glucagon increased flux through phenylalanine hydroxylase; a half-maximal response was obtained at 0.7 nM. Under control conditions, 0.2-0.3 mol of phosphate were incorporated per mol of subunit of the hydroxylase at steady state. Glucagon increased this incorporation of phosphate into the hydroxylase to a maximal value of approx. 0.6 mol of phosphate per subunit; a half-maximal response was obtained at 0.3 nM. Glucagon, added simultaneously with [32P]Pi to liver cells, inhibited incorporation of 32P into the enzyme. The effects of glucagon were reproduced with dibutyryl cyclic AMP. Changes in phosphorylation correlated closely with changes in flux through phenylalanine hydroxylase in cell incubations.

SUBMITTER: Fisher MJ

PROVIDER: S-EPMC1153450 | biostudies-other | 1984 Apr

REPOSITORIES: biostudies-other

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Phosphorylation of Phenylalanine Hydroxylase Increases the Rate Constant for Formation of the Activated Conformation of the Enzyme.

Project description:Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that is activated by phenylalanine. The enzyme is also phosphorylated by protein kinase A, but the effects of phosphorylation are unclear. Recent structural studies ( Meisburger et al. ( 2016 ) J. Amer. Chem. Soc. 138 , 6506 - 6516 ) support a model in which activation of the enzyme involves dimerization of the regulatory domains, creating the allosteric site for phenylalanine at the dimer interface. This conformational change also results in a change in the fluorescence of the protein that can be used to monitor activation. The kinetics of activation of PheH are biphasic over a range of phenylalanine concentrations. These data are well-described by a model involving an initial equilibrium between the resting form and the activated conformation, with a value of the equilibrium constant for formation of the activated conformation, L, equal to 0.007, followed by binding of two molecules of phenylalanine. Phosphorylation increases L 10-fold by increasing the rate constant for conversion of the resting form to the activated form. The results provide functional support for the previous structural model, identify the specific effect of phosphorylation on the enzyme, and rationalize the lack of change in the protein structure upon phosphorylation.

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The polyamine-dependent modulation of phenylalanine hydroxylase phosphorylation state and enzymic activity in isolated liver cells.

Project description:The role of polyamines in the control of phenylalanine hydroxylase phosphorylation state and enzymic activity was investigated. Pre-treatment of liver cells with spermine (1 mM) abolishes the glucagon (1 nM)-stimulated increase in hydroxylase phosphorylation. Concurrently there is a decrease in phenylalanine hydroxylation flux, reflecting decreased enzyme activity; 50% inhibition occurs at approx. 10 microM-spermine. These results are discussed in the context of reports concerning the properties of protein phosphatase 2A.

| S-EPMC1146977 | biostudies-other

Structural and stability effects of phosphorylation: Localized structural changes in phenylalanine hydroxylase.

Project description:Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cAMP-dependent protein kinase increases the basal activity of the enzyme and its resistance to tryptic proteolysis. The modeled structures of the full-length phosphorylated and unphosphorylated enzyme were subjected to molecular dynamics simulations, and we analyzed the energy of charge-charge interactions for individual ionizable residues in the final structures. These calculations showed that the conformational changes induced by incorporation of phosphate were localized and limited mostly to the region around the phosphoserine (Arg13-Asp17) and a region around the active site in the catalytic domain that includes residues involved in the binding of the iron and the substrate L-Phe (Arg270 and His285). The absence of a generalized conformational change was confirmed by differential scanning calorimetry, thermal-dependent circular dichroism, fluorescence spectroscopy, and limited chymotryptic proteolysis of the phosphorylated and unphosphorylated PAH. Our results explain the effect of phosphorylation of PAH on both the resistance to proteolysis specifically by trypsin-like enzymes and on the increase in catalytic efficiency.

| S-EPMC2286772 | biostudies-literature

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Project description:The plasma concentration of phenylalanine and tyrosine decreases in normal rats during the first few postnatal days; subsequently, the concentration of phenylalanine remains more or less constant, whereas that of tyrosine exhibits a high peak on day 13. The basal concentrations of the two amino acids were not altered by injections of thyroxine or cortisol, except in 13-day-old rats, when an injection of cortisol decreased the concentration of tyrosine. In young rats (13-15 days old), treatment with cortisol increased the activity of phenylalanine hydroxylase in the liver (measured in vitro) and accelerated the metabolism of administered phenylalanine: the rate constant of the disappearance of phenylalanine from plasma and the initial increase in tyrosine in plasma correlated quantitatively with the activity of phenylalanine hydroxylase in the liver. In adult rats, the inhibition of this enzyme (attested by assay in vitro) by p-chlorophenylalanine resulted in a proportionate decrease in tyrosine formation from an injection of phenylalanine. However, the quantitative relationship between liver phenylalanine hydroxylase activity and phenylalanine metabolism within the group of young rats was different from that observed among adult rats.

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Experimental determination of the phosphorylation state of phenylalanine hydroxylase.

Project description:A monoclonal antibody (PH 7), which recognizes the phosphorylated form of phenylalanine hydroxylase from human liver, has been used for the analysis of the enzyme in crude cell extracts from rat. In immunoblot analyses of rat liver cell extracts, the extent of binding of PH 7 closely correlates with the phosphorylation state of phenylalanine hydroxylase, as judged by [32P]Pi incorporation. These observations have made possible the rapid non-radioactive quantification of hormonal effects on phenylalanine hydroxylase phosphorylation state. In particular, the glucagon-dependent phosphorylation of phenylalanine hydroxylase in liver cells was investigated. Epidermal growth factor was shown to modulate this process. In addition, this technique was used to demonstrate, for the first time, that dibutyryl cyclic AMP, unlike the Ca2+ ionophore A23187, stimulates the phosphorylation of phenylalanine hydroxylase in isolated kidney tubules from rat.

| S-EPMC1136920 | biostudies-other

Deubiquitinase USP19 extends the residual enzymatic activity of phenylalanine hydroxylase variants.

Project description:Phenylalanine hydroxylase (PAH) is a key enzyme in mammals that maintains the phenylalanine (Phe) concentration at an appropriate physiological level. Some genetic mutations in the PAH gene lead to destabilization of the PAH enzyme, leading to phenylketonuria (PKU). Destabilized PAH variants can have a certain amount of residual enzymatic activity that is sufficient for metabolism of Phe. However, accelerated degradation of those variants can lead to insufficient amounts of cellular PAH protein. The optimal protein level of PAH in cells is regulated by a balancing act between E3 ligases and deubiquitinating enzymes (DUBs). In this work, we analyzed the protein expression and stability of two PKU-linked PAH protein variants, R241C and R243Q, prevalent in the Asian population. We found that the tested PAH variants were highly ubiquitinated and thus targeted for rapid protein degradation. We demonstrated that USP19, a DUB that interacts with both PAH variants, plays a regulatory role by extending their half-lives. The deubiquitinating activity of USP19 prevents protein degradation and increases the abundance of both PAH protein variants. Thus, our study reveals a novel mechanism by which deubiquitinating activity of USP19 extends the residual enzymatic activity of PAH variants.

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Mutagenesis of a specificity-determining residue in tyrosine hydroxylase establishes that the enzyme is a robust phenylalanine hydroxylase but a fragile tyrosine hydroxylase.

Project description:The aromatic amino acid hydroxylases tyrosine hydroxylase (TyrH) and phenylalanine hydroxylase (PheH) have essentially identical active sites; however, PheH is nearly incapable of hydroxylating tyrosine, while TyrH can readily hydroxylate both tyrosine and phenylalanine. Previous studies have indicated that Asp425 of TyrH is important in determining the substrate specificity of that enzyme [Daubner, S. C., Melendez, J., and Fitzpatrick, P. F. (2000) Biochemistry 39, 9652-9661]. Alanine-scanning mutagenesis of amino acids 423-427, a mobile loop containing Asp425, shows that only mutagenesis of Asp425 alters the activity of the enzyme significantly. Saturation mutagenesis of Asp425 results in large (up to 10(4)) decreases in the V(max) and V(max)/K(tyr) values for tyrosine hydroxylation, but only small decreases or even increases in the V(max) and V(max)/K(phe) values for phenylalanine hydroxylation. The decrease in the tyrosine hydroxylation activity of the mutant proteins is due to an uncoupling of tetrahydropterin oxidation from amino acid hydroxylation with tyrosine as the amino acid substrate. In contrast, with the exception of the D425W mutant, the extent of coupling of tetrahydropterin oxidation and amino acid hydroxylation is unaffected or increases with phenylalanine as the amino acid substrate. The decrease in the V(max) value with tyrosine as the substrate shows a negative correlation with the hydrophobicity of the amino acid residue at position 425. The results are consistent with a critical role of Asp425 being to prevent a hydrophobic interaction that results in a restricted active site in which hydroxylation of tyrosine does not occur.

| S-EPMC3584195 | biostudies-literature

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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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Regulation of phenylalanine hydroxylase: conformational changes upon phosphorylation detected by H/D exchange and mass spectrometry.

Project description:The enzyme phenylalanine hydroxylase catalyzes the hydroxylation of excess phenylalanine in the liver to tyrosine. The enzyme is regulated allosterically by phenylalanine and by phosphorylation of Ser16. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into any structural change upon phosphorylation. Peptides in both the catalytic and regulatory domains show increased deuterium incorporation into the phosphorylated protein. Deuterium is incorporated into fewer peptides than when the enzyme is activated by phenylalanine, and the incorporation is slower. This establishes that the conformational change upon phosphorylation of phenylalanine hydroxylase is different from and less extensive than that upon phenylalanine activation.

| S-EPMC3683384 | biostudies-literature

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.

| S-EPMC3664513 | biostudies-literature

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