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Torgeir Flatmark - One of the best experts on this subject based on the ideXlab platform.
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Possible Contributions of Labile Asparagine Residues to Differences in Regulatory Properties of Human and Rat Phenylalanine Hydroxylase
Chemistry and Biology of Pteridines and Folates, 2020Co-Authors: Raquel Negrão Carvalho, Therese Solstad, N. E. Robinson, A. B. Robinson, Torgeir FlatmarkAbstract:Phenylalanine hydroxylase (PAH, Phenylalanine 4-Monooxygenase, EC 1.14.16.1) catalyzes a single hydroxylation by an intermolecular oxygenation reaction of an aromatic ring (L-Phenylalanine, L-Phe) in an Fe(II)- and pterin-dependent fashion (reviewed in (1,2)). X-ray crystal structure analyses of human and rat PAH (hPAH and rPAH) have revealed that the monomer is organized into three main domains, i.e. a N-terminal regulatory domain, a catalytic domain and a small C-terminal dimerization/tetramerization domain (reviewed in (2)). PAH isolated from rat liver and as recombinant human enzyme are both in a tetramer dimer equilibrium (3,4). The tetrameric form is a dimer of dimers with defined interphases between the subunits in the dimers. The 41 C-terminal residues that comprise the tetramerization motif are organized into an “arm” consisting of two short s strands, forming a s ribbon, and a 40-A long α helix (Gln428-Lys452)(5). The helices interact with each other, forming an anti-parallel coiled coil structure in the center of the tetramers and mutually switch their position to promote tetramerization (5). A comparison of the crystal structure of hPAH (5,6) and rPAH (7) have revealed the same overall fold for the highly conserved catalytic domain of the two enzyme forms. Thus, the human and rat enzyme share 96% sequence homology and have several molecular and kinetic properties in common (8).
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Studies on the regulatory properties of the pterin cofactor and dopamine bound at the active site of human Phenylalanine hydroxylase.
FEBS Journal, 2003Co-Authors: Therese Solstad, Anne Jorunn Stokka, O.a. Andersen, Torgeir FlatmarkAbstract:The catalytic activity of Phenylalanine hydroxylase (PAH, Phenylalanine 4-Monooxygenase EC 1.14.16.1) is regulated by three main mechanisms, i.e. substrate (l-Phenylalanine, L-Phe) activation, pterin cofactor inhibition and phosphorylation of a single serine (Ser16) residue. To address the molecular basis for the inhibition by the natural cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, its effects on the recombinant tetrameric human enzyme (wt-hPAH) was studied using three different conformational probes, i.e. the limited proteolysis by trypsin, the reversible global conformational transition (hysteresis) triggered by L-Phe binding, as measured in real time by surface plasmon resonance analysis, and the rate of phosphorylation of Ser16 by cAMP-dependent protein kinase. Comparison of the inhibitory properties of the natural cofactor with the available three-dimensional crystal structure information on the ligand-free, the binary and the ternary complexes, have provided important clues concerning the molecular mechanism for the negative modulatory effects. In the binary complex, the binding of the cofactor at the active site results in the formation of stabilizing hydrogen bonds between the dihydroxypropyl side-chain and the carbonyl oxygen of Ser23 in the autoregulatory sequence. L-Phe binding triggers local as well as global conformational changes of the protomer resulting in a displacement of the cofactor bound at the active site by 2.6 A (mean distance) in the direction of the iron and Glu286 which causes a loss of the stabilizing hydrogen bonds present in the binary complex and thereby a complete reversal of the pterin cofactor as a negative effector. The negative modulatory properties of the inhibitor dopamine, bound by bidentate coordination to the active site iron, is explained by a similar molecular mechanism including its reversal by substrate binding. Although the pterin cofactor and the substrate bind at distinctly different sites, the local conformational changes imposed by their binding at the active site have a mutual effect on their respective binding affinities.
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A comparison of kinetic and regulatory properties of the tetrameric and dimeric forms of wild‐type and Thr427→Pro mutant human Phenylalanine hydroxylase
FEBS Journal, 2001Co-Authors: Elisa Bjørgo, Raquel Negrão Carvalho, Torgeir FlatmarkAbstract:Recombinant human Phenylalanine hydroxylase (hPAH, Phenylalanine 4-Monooxygenase EC 1.14.16.1) is catalytically active both as a tetramer and a dimer [Knappskog, P.M., Flatmark, T., Aarden, J.M., Haavik, J. and Martinez, A. (1996) Eur. J. Biochem.242, 813–821]. In the present study we have further characterized the differences in kinetic and regulatory properties of the two oligomeric forms when expressed in Escherichia coli. The positive cooperativity of l-Phe binding to the tetrameric form both in enzyme kinetic studies (h = 1.6) and intrinsic tryptophan fluorescence measurements (h = 2.3) was abolished in the dimer, which also revealed a catalytic efficiency (Vmax/[S]0.5) of only 35% of the tetramer. Whereas the catalytic activity of the tetramer was activated fivefold to sixfold by preincubation with l-Phe, the dimer revealed only a 1.6-fold activation. The crystal structure has identified a five-residue flexible hinge region (Asp425–Gln429) that links the β-strand Tβ2 (Ile421–Leu424) and the 24 residue amphipathic α-helix Tα1 (Gln428–Lys452) at the C-terminus which forms an antiparallel coiled-coil structure in the center of the tetramer [Fusetti, F., Erlandsen, H., Flatmark, T. & Stevens, R.C. (1998) J. Biol. Chem.273, 16962–16967]. The potential role of this flexible hinge in the tetramerization and the conformational transition of wt-hPAH on the cooperative binding of l-Phe was examined by site-specific mutagenesis. Substitution of Thr427 by a Pro (as in tyrosine hydroxylase) resulted in a mutant protein which was isolated mainly (about 95%) as a dimer. The isolated tetramer of T427P revealed no kinetic cooperativity of l-Phe binding, the catalytic efficiency (Vmax/[S]0.5) was decreased to about 39% of the wild-type tetramer and it was not activated by l-Phe preincubation. The dimeric forms of T427P and wt-hPAH revealed rather similar kinetic properties. The lack of kinetic cooperativity of the T427P tetramer was associated with a corresponding change in the binding isotherm for l-Phe as studied by intrinsic tryptophan fluorescence measurements. Protein stability was also reduced both for the E. coli expressed and the in vitro synthesized mutant enzyme. Collectively, these results indicate that the positive cooperativity of l-Phe binding to wt-hPAH requires a tetrameric enzyme with a C-terminal flexibel hinge region (Asp425–Gln429) which has a structural role in the formation of the enzyme tetramer. Furthermore, this hinge region represents a motif in the PAH structure that is involved in the conformational change transmitted through the protein on the cooperative binding of l-Phe to tetrameric wt-hPAH. This conclusion is further supported by studies on two disease (phenylketonuria)-associated mutant forms.
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Crystallization and preliminary diffraction analysis of a truncated homodimer of human Phenylalanine hydroxylase
FEBS Letters, 1997Co-Authors: H Erlandsen, Aurora Martinez, Jan Haavik, P M Knappskog, E Hough, Torgeir FlatmarkAbstract:Abstract A recombinant truncated form (Δ1-102/Δ428-452) of the non-heme iron-dependent metalloenzyme human Phenylalanine hydroxylase (hPAH, Phenylalanine 4-Monooxygenase; EC 1.14.16.1) was expressed in E. coli, purified to homogeneity as a homodimer (70 kDa) and crystallized using the hanging drop vapour diffusion method. The crystals are orthorhombic, space group C222 with cell dimensions of a=66.6 A, b=108.4 A, c=125.7 A. The calculated packing parameter (Vm) is 3.24 A3/Da with four 2-fold symmetric dimers (or eight momomers) in the unit cell. Data have been collected to 2.0 A resolution.
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Expression of wild type and mutant forms of human Phenylalanine hydroxylase in E. coli.
Advances in Experimental Medicine and Biology, 1993Co-Authors: P M Knappskog, Torgeir Flatmark, Aurora Martinez, Jan Haavik, Sigridur Olafsdottir, Hans Geir Eiken, Jaran ApoldAbstract:Phenylketonuria (PKU) is an autosomal recessive disease caused by the absence or severely reduced enzymatic activity of the hepatic enzyme Phenylalanine hydroxylase (Phenylalanine 4-Monooxygenase, EC 1.14.16.1, PAH). The loss of enzymatic activity found in PKU patients is a result of single base substitutions or small deletions in the PAH gene. Presently, more than 70 different mutations associated with the disease are known. PKU and non-PKU hyperPhenylalaninemia (HPA) patients show extensive clinical heterogeneity. To understand the molecular mechanism of this heterogeneity, it is necessary to characterize the structural and functional properties of the different mutant PAH forms. Liver biopsies from PKU/HPA patients are usually not available, but in vitro expression in eukaryotic cells has been used for the characterization of certain mutant forms. However, it has been a problem to express stable forms of PAH in such in vitro systems1, and therefore only a few mutant forms have been further characterized so far. This led us to clone and express the normal and several mutated forms of human PAH cDNA into the pET-plasmid system of E. coli (Novagen). Here we report the expression of both normal and seven disease associated forms of PAH, i.e. mutations in exon 7 and 8 of the PAH gene.
Glyn B Steventon - One of the best experts on this subject based on the ideXlab platform.
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Phenylalanine 4 Monooxygenase the sulfoxidation polymorphism
Xenobiotica, 2020Co-Authors: S C Mitchell, Glyn B SteventonAbstract:Abstract1. Consistent differences in the proportion of an orally administered dose of S-carboxymethyl-l-cysteine subsequently excreted in the urine as S-oxide metabolites were reported 40 years ago...
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post translational activation of human Phenylalanine 4 Monooxygenase from an endobiotic to a xenobiotic enzyme by reactive oxygen and reactive nitrogen species
Xenobiotica, 2010Co-Authors: A Antypa, S C Mitchell, C Rebello, A Biernacka, K Krajewski, J Cassam, Glyn B SteventonAbstract:An investigation into the post-translational activation of cDNA-expressed human Phenylalanine 4-Monooxygenase and human hepatic cytosolic fraction Phenylalanine 4-Monooxygenase activity with respect to both endobiotic metabolism and xenobiotic metabolism revealed that the reactive oxygen species (hydrogen peroxide and hydroxyl radical) and reactive nitrogen species (nitric oxide and peroxynitrite) could elicit the post-translational activation of the enzyme with respect to both of these biotransformation reactions.In virtually all instances, the Km values were decreased and the Vmax values were increased; the only exceptions observed being with hydrogen peroxide and L-Phenylalanine.These effects were shown to occur at activator concentrations known to exist in physiological situations and, hence, suggest that reactive oxygen and reactive nitrogen species may cause, and may be involved with, the post-translational activation of Phenylalanine 4-Monooxygenase within the human body.This mechanism, in response...
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Phenylalanine 4 Monooxygenase and the role of endobiotic metabolism enzymes in xenobiotic biotransformation
Expert Opinion on Drug Metabolism & Toxicology, 2009Co-Authors: Glyn B Steventon, S C MitchellAbstract:Phenylalanine 4-Monooxygenase is the key enzyme in the sulfoxidation of the thioether drug S-carboxymethyl-l-cysteine and its thioether metabolites, S-methyl-l-cysteine, N-acetyl-S-carboxymethyl-l-cysteine and N-acetyl-S-methyl-l-cysteine in humans, and a number of other mammalian species. The kinetics constants of the sulfoxidation reaction (Km, Vmax and CLE) have been investigated in cytosolic fractions derived from rat and human liver, in cytosolic fractions of HepG2 cells and using both human and mouse cDNA expressed Phenylalanine 4-Monooxygenase. Differences in Km, Vmax and CLE of S-carboxymethyl-l-cysteine have been seen in HepG2 cells and human and mouse cDNA expressed Phenylalanine 4-Monooxygenase when compared to both rat and human hepatic cytosolic fractions. The association of the genetic polymorphism in the sulfoxidation of S-carboxymethyl-l-cysteine is highlighted with particular reference to this biotransformation reaction as being a biomarker of disease susceptibility in Parkinson's, Alzhei...
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enzyme kinetic and molecular modelling studies of sulphur containing substrates of Phenylalanine 4 Monooxygenase
Journal of Enzyme Inhibition and Medicinal Chemistry, 2008Co-Authors: N G Patel, S C Mitchell, Boontarika Boonyapiwat, Ben Forbes, C Iliadou, David J Barlow, Glyn B SteventonAbstract:Previous investigations into the binding of substrates/cofactors to the PAH active site have only concentrated on Phe, thienylalanine and BH4. This is the first reported investigation to model aliphatic thioether amino acid substrates to PAH. The clearance of the thioether substrates (4.82-79.09% of Phe) in the rat and human (1.19-37.41% of Phe) showed species differences. The xenobiotic thioether substrates (SMC and SCMC) were predicted to be poor substrates for PAH by the molecular modelling investigation and this has now been confirmed by the in vitro enzyme kinetic data. However, reaction phenotyping investigations have found that PAH was the major enzyme involved in the metabolism of SCMC in vitro and in vivo.
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Phenylalanine 4 Monooxygenase and the s oxidation of s carboxymethyl l cysteine by human cytosolic fractions
Drug metabolism and drug interactions, 2008Co-Authors: Boontarika Boonyapiwat, S C Mitchell, Ben Forbes, Glyn B SteventonAbstract:: The purpose of this investigation was to reaction phenotype the identity of the cytosolic enzyme responsible for the S-oxidation of S-carboxymethyl-L-cysteine (SCMC) in female human hepatic cytosolic fractions. The identity of this enzyme in the female Wistar rat hepatic cytosolic fraction was found to be Phenylalanine 4-Monooxygenase (PAH). In pooled female human hepatic cytosolic fractions the calculated K(m) and V(max) for substrate (SCMC) activated PAH was 16.22 +/- 11.31 mM and 0.87 +/- 0.41 nmoles x min(-1) mg(-1). The experimental data modelled to the Michaelis-Menten equation with noncompetitive substrate inhibition. When the cytosolic fractions were activated with lysophophatidylcholine the V(max) increased to 52.31 +/- 11.72 nmoles x min(-1) mg(-1) but the K(m) remained unchanged at 16.53 +/- 2.32 mM. A linear correlation was seen in the production of Tyr and SCMC R/S S-oxide in 20 individual female hepatic cytosolic fractions for both substrate and lysophosphatidylcholine activated PAH (r(s) > 0.96). Inhibitor studies found that the specific chemical and antibody inhibitors of PAH reduced the production of Tyr and SCMC R/S S-oxide in these in vitro PAH assays. An investigation of the mechanism of interaction of SCMC with PAH indicated that the drug was a competitive inhibitor of the aromatic C-oxidation of Phe with a calculated K(i) of 17.23 +/- 4.15 mM. The requirement of BH4 as cofactor and the lack of effect of the specific tyrosine hydroxylase, tryptophan hydroxylase and nitric oxide synthase inhibitors on the S-oxidation of SCMC all indicate that PAH was the enzyme responsible for this biotransformation reaction in human hepatic cytosolic fractions.
S C Mitchell - One of the best experts on this subject based on the ideXlab platform.
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Phenylalanine 4 Monooxygenase the sulfoxidation polymorphism
Xenobiotica, 2020Co-Authors: S C Mitchell, Glyn B SteventonAbstract:Abstract1. Consistent differences in the proportion of an orally administered dose of S-carboxymethyl-l-cysteine subsequently excreted in the urine as S-oxide metabolites were reported 40 years ago...
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post translational activation of human Phenylalanine 4 Monooxygenase from an endobiotic to a xenobiotic enzyme by reactive oxygen and reactive nitrogen species
Xenobiotica, 2010Co-Authors: A Antypa, S C Mitchell, C Rebello, A Biernacka, K Krajewski, J Cassam, Glyn B SteventonAbstract:An investigation into the post-translational activation of cDNA-expressed human Phenylalanine 4-Monooxygenase and human hepatic cytosolic fraction Phenylalanine 4-Monooxygenase activity with respect to both endobiotic metabolism and xenobiotic metabolism revealed that the reactive oxygen species (hydrogen peroxide and hydroxyl radical) and reactive nitrogen species (nitric oxide and peroxynitrite) could elicit the post-translational activation of the enzyme with respect to both of these biotransformation reactions.In virtually all instances, the Km values were decreased and the Vmax values were increased; the only exceptions observed being with hydrogen peroxide and L-Phenylalanine.These effects were shown to occur at activator concentrations known to exist in physiological situations and, hence, suggest that reactive oxygen and reactive nitrogen species may cause, and may be involved with, the post-translational activation of Phenylalanine 4-Monooxygenase within the human body.This mechanism, in response...
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Phenylalanine 4 Monooxygenase and the role of endobiotic metabolism enzymes in xenobiotic biotransformation
Expert Opinion on Drug Metabolism & Toxicology, 2009Co-Authors: Glyn B Steventon, S C MitchellAbstract:Phenylalanine 4-Monooxygenase is the key enzyme in the sulfoxidation of the thioether drug S-carboxymethyl-l-cysteine and its thioether metabolites, S-methyl-l-cysteine, N-acetyl-S-carboxymethyl-l-cysteine and N-acetyl-S-methyl-l-cysteine in humans, and a number of other mammalian species. The kinetics constants of the sulfoxidation reaction (Km, Vmax and CLE) have been investigated in cytosolic fractions derived from rat and human liver, in cytosolic fractions of HepG2 cells and using both human and mouse cDNA expressed Phenylalanine 4-Monooxygenase. Differences in Km, Vmax and CLE of S-carboxymethyl-l-cysteine have been seen in HepG2 cells and human and mouse cDNA expressed Phenylalanine 4-Monooxygenase when compared to both rat and human hepatic cytosolic fractions. The association of the genetic polymorphism in the sulfoxidation of S-carboxymethyl-l-cysteine is highlighted with particular reference to this biotransformation reaction as being a biomarker of disease susceptibility in Parkinson's, Alzhei...
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enzyme kinetic and molecular modelling studies of sulphur containing substrates of Phenylalanine 4 Monooxygenase
Journal of Enzyme Inhibition and Medicinal Chemistry, 2008Co-Authors: N G Patel, S C Mitchell, Boontarika Boonyapiwat, Ben Forbes, C Iliadou, David J Barlow, Glyn B SteventonAbstract:Previous investigations into the binding of substrates/cofactors to the PAH active site have only concentrated on Phe, thienylalanine and BH4. This is the first reported investigation to model aliphatic thioether amino acid substrates to PAH. The clearance of the thioether substrates (4.82-79.09% of Phe) in the rat and human (1.19-37.41% of Phe) showed species differences. The xenobiotic thioether substrates (SMC and SCMC) were predicted to be poor substrates for PAH by the molecular modelling investigation and this has now been confirmed by the in vitro enzyme kinetic data. However, reaction phenotyping investigations have found that PAH was the major enzyme involved in the metabolism of SCMC in vitro and in vivo.
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Phenylalanine 4 Monooxygenase and the s oxidation of s carboxymethyl l cysteine by human cytosolic fractions
Drug metabolism and drug interactions, 2008Co-Authors: Boontarika Boonyapiwat, S C Mitchell, Ben Forbes, Glyn B SteventonAbstract:: The purpose of this investigation was to reaction phenotype the identity of the cytosolic enzyme responsible for the S-oxidation of S-carboxymethyl-L-cysteine (SCMC) in female human hepatic cytosolic fractions. The identity of this enzyme in the female Wistar rat hepatic cytosolic fraction was found to be Phenylalanine 4-Monooxygenase (PAH). In pooled female human hepatic cytosolic fractions the calculated K(m) and V(max) for substrate (SCMC) activated PAH was 16.22 +/- 11.31 mM and 0.87 +/- 0.41 nmoles x min(-1) mg(-1). The experimental data modelled to the Michaelis-Menten equation with noncompetitive substrate inhibition. When the cytosolic fractions were activated with lysophophatidylcholine the V(max) increased to 52.31 +/- 11.72 nmoles x min(-1) mg(-1) but the K(m) remained unchanged at 16.53 +/- 2.32 mM. A linear correlation was seen in the production of Tyr and SCMC R/S S-oxide in 20 individual female hepatic cytosolic fractions for both substrate and lysophosphatidylcholine activated PAH (r(s) > 0.96). Inhibitor studies found that the specific chemical and antibody inhibitors of PAH reduced the production of Tyr and SCMC R/S S-oxide in these in vitro PAH assays. An investigation of the mechanism of interaction of SCMC with PAH indicated that the drug was a competitive inhibitor of the aromatic C-oxidation of Phe with a calculated K(i) of 17.23 +/- 4.15 mM. The requirement of BH4 as cofactor and the lack of effect of the specific tyrosine hydroxylase, tryptophan hydroxylase and nitric oxide synthase inhibitors on the S-oxidation of SCMC all indicate that PAH was the enzyme responsible for this biotransformation reaction in human hepatic cytosolic fractions.
Aurora Martinez - One of the best experts on this subject based on the ideXlab platform.
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Crystallization and preliminary diffraction analysis of a truncated homodimer of human Phenylalanine hydroxylase
FEBS Letters, 1997Co-Authors: H Erlandsen, Aurora Martinez, Jan Haavik, P M Knappskog, E Hough, Torgeir FlatmarkAbstract:Abstract A recombinant truncated form (Δ1-102/Δ428-452) of the non-heme iron-dependent metalloenzyme human Phenylalanine hydroxylase (hPAH, Phenylalanine 4-Monooxygenase; EC 1.14.16.1) was expressed in E. coli, purified to homogeneity as a homodimer (70 kDa) and crystallized using the hanging drop vapour diffusion method. The crystals are orthorhombic, space group C222 with cell dimensions of a=66.6 A, b=108.4 A, c=125.7 A. The calculated packing parameter (Vm) is 3.24 A3/Da with four 2-fold symmetric dimers (or eight momomers) in the unit cell. Data have been collected to 2.0 A resolution.
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Expression of wild type and mutant forms of human Phenylalanine hydroxylase in E. coli.
Advances in Experimental Medicine and Biology, 1993Co-Authors: P M Knappskog, Torgeir Flatmark, Aurora Martinez, Jan Haavik, Sigridur Olafsdottir, Hans Geir Eiken, Jaran ApoldAbstract:Phenylketonuria (PKU) is an autosomal recessive disease caused by the absence or severely reduced enzymatic activity of the hepatic enzyme Phenylalanine hydroxylase (Phenylalanine 4-Monooxygenase, EC 1.14.16.1, PAH). The loss of enzymatic activity found in PKU patients is a result of single base substitutions or small deletions in the PAH gene. Presently, more than 70 different mutations associated with the disease are known. PKU and non-PKU hyperPhenylalaninemia (HPA) patients show extensive clinical heterogeneity. To understand the molecular mechanism of this heterogeneity, it is necessary to characterize the structural and functional properties of the different mutant PAH forms. Liver biopsies from PKU/HPA patients are usually not available, but in vitro expression in eukaryotic cells has been used for the characterization of certain mutant forms. However, it has been a problem to express stable forms of PAH in such in vitro systems1, and therefore only a few mutant forms have been further characterized so far. This led us to clone and express the normal and several mutated forms of human PAH cDNA into the pET-plasmid system of E. coli (Novagen). Here we report the expression of both normal and seven disease associated forms of PAH, i.e. mutations in exon 7 and 8 of the PAH gene.
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the cooperative binding of Phenylalanine to Phenylalanine 4 Monooxygenase studied by 1h nmr paramagnetic relaxation changes in water accessibility to the iron at the active site upon substrate binding
FEBS Journal, 1993Co-Authors: Aurora Martinez, Sigridur Olafsdottir, Torgeir FlatmarkAbstract:The effect of the paramagnetic high-spin Fe(III) ion in Phenylalanine 4-Monooxygenase (Phenylalanine hydroxylase, EC 1.14.16.1) on the water proton longitudinal relaxation rate has been used to study the environment of the iron center. The relaxation rate was measured as a function of the concentration of enzyme, substrate (Phenylalanine), inhibitor (noradrenaline) and activator (lysolecithin), as well as of the temperature (18–40°C) and the external magnetic field strength (100–600 MHz). From the frequency dependence of the relaxation rate, an effective correlation time (τc) of 4.2(± 0.5) × 10−10 s was calculated for the enzyme-substrate complex, which most likely represents the electron spin relaxation rate (τs) for Fe(III) (S= 5/2) in this complex. The relaxation rate was proportional to the concentration of enzyme (0.04–1 mM) both in the absence and presence of Phenylalanine, but the paramagnetic molar relaxivity at 400 MHz and 22°C decreased from 2.2(± 0.05) × 103 s–1· M–1 in the enzyme as isolated to 1.2(± 0.06) × 103 s–1· M–1 in the presence of saturating concentrations of the substrate. The activation energy of the relaxation rate also decreased from 11.3 ± 0.8 kJ/mol to –1.5 ± 0.2 kJ/mol upon incubation of the enzyme with 5 mM Phenylalanine. The results obtained can be interpreted in terms of a slowly exchanging water molecule coordinated to the catalytic paramagnetic Fe(III) in the native and resting enzyme, and that this water molecule seems to be displaced from coordination on the binding of substrate or inhibitor. Moreover, the effect of increasing concentrations of Phenylalanine and noradrenaline on the water proton relaxation rate and on the hydrophobic surface properties of the enzyme indicate that substrate and inhibitor induce a similar cooperative conformational change upon binding at the active site. By contrast, the activator lysolecithin does not seem to affect the interaction of water with the catalytic Fe(III).
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Cooperative homotropic interaction of l‐noradrenaline with the catalytic site of Phenylalanine 4‐Monooxygenase
FEBS Journal, 1990Co-Authors: Aurora Martinez, Jan Haavik, Torgeir FlatmarkAbstract:Catecholamines (adrenaline, noradrenaline and dopamine) are potent inhibitors of Phenylalanine 4-Monooxygenase (Phenylalanine hydroxylase, EC 1.14.16.1). The amines bind to the enzyme by a direct coordination to the high-spin (S= 5/2) Fe(III) at the active site (charge transfer interaction), as seen by resonance Raman and EPR spectroscopy. Experimental evidence is presented that a group with an apparent pKa value of about 5.1 (20°C) is involved in the interaction between the catecholamine and the enzyme. The high-affinity binding of l-noradrenaline to Phenylalanine hydroxylase, as studied by equilibrium microdialysis (anaerobically) and ultrafiltration (aerobically), shows positive cooperativity (h= 1.9); at pH 7.2 and 20°C the rat enzyme binds about 0.5 mol l-noradrenaline/mol subunit with a half-maximal binding (S50) at 0.25 μM l-noradrenaline. No binding to the ferrous form of the enzyme was observed. The affinity decreases with decreasing pH, by phosphorylation and by preincubation of the enzyme with the substrate l-Phenylalanine, while it increases after alkylation of the enzyme with the activator N-ethylmaleimide. Preincubation of the enzyme with l-Phenylalanine also leads to a complete loss of the cooperativity of l-noradrenaline binding (h= 1.0). The many similarities in binding properties of the inhibitor l-noradrenaline and the activator/substrate l-Phenylalanine makes it likely that the cooperative interactions of these effectors are due to their binding to the same site. The high-affinity of catecholamines to Phenylalanine hydroxylase is a valuable probe to study the active site of this enzyme and is also relevant for the homologous enzyme tyrosine hydroxylase, which is purified as a stable catecholamine-Fe(III) complex.
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cooperative homotropic interaction of l noradrenaline with the catalytic site of Phenylalanine 4 Monooxygenase
FEBS Journal, 1990Co-Authors: Aurora Martinez, Jan Haavik, Torgeir FlatmarkAbstract:Catecholamines (adrenaline, noradrenaline and dopamine) are potent inhibitors of Phenylalanine 4-Monooxygenase (Phenylalanine hydroxylase, EC 1.14.16.1). The amines bind to the enzyme by a direct coordination to the high-spin (S= 5/2) Fe(III) at the active site (charge transfer interaction), as seen by resonance Raman and EPR spectroscopy. Experimental evidence is presented that a group with an apparent pKa value of about 5.1 (20°C) is involved in the interaction between the catecholamine and the enzyme. The high-affinity binding of l-noradrenaline to Phenylalanine hydroxylase, as studied by equilibrium microdialysis (anaerobically) and ultrafiltration (aerobically), shows positive cooperativity (h= 1.9); at pH 7.2 and 20°C the rat enzyme binds about 0.5 mol l-noradrenaline/mol subunit with a half-maximal binding (S50) at 0.25 μM l-noradrenaline. No binding to the ferrous form of the enzyme was observed. The affinity decreases with decreasing pH, by phosphorylation and by preincubation of the enzyme with the substrate l-Phenylalanine, while it increases after alkylation of the enzyme with the activator N-ethylmaleimide. Preincubation of the enzyme with l-Phenylalanine also leads to a complete loss of the cooperativity of l-noradrenaline binding (h= 1.0). The many similarities in binding properties of the inhibitor l-noradrenaline and the activator/substrate l-Phenylalanine makes it likely that the cooperative interactions of these effectors are due to their binding to the same site. The high-affinity of catecholamines to Phenylalanine hydroxylase is a valuable probe to study the active site of this enzyme and is also relevant for the homologous enzyme tyrosine hydroxylase, which is purified as a stable catecholamine-Fe(III) complex.
Stein Ove Doskeland - One of the best experts on this subject based on the ideXlab platform.
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Phenylalanine positively modulates the camp dependent phosphorylation and negatively modulates the vasopressin induced and okadaic acid induced phosphorylation of Phenylalanine 4 Monooxygenase in intact rat hepatocytes
FEBS Journal, 1992Co-Authors: Anne P Doskeland, Torgeir Flatmark, Olav Karsten Vintermyr, Richard G H Cotton, Stein Ove DoskelandAbstract:The state of phosphorylation of Phenylalanine hydroxylase was determined in isolated intact rat hepatocytes. 32P-labeled Phenylalanine hydroxylase was immunoisolated from cells loaded with 32Pi or from cell extracts ‘back-phosphorylated’ with [γ-32P]ATP by cAMP-dependent protein kinase. The rate of Phenylalanine hydroxylase phosphorylation in cells with elevated cAMP was similar to that observed for the isolated enzyme phosphorylated by homogeneous cAMP-dependent protein kinase. The phosphorylation rate in cAMP-stimulated cells was increased up to four times (reaching 0.018 s–1) by the presence of Phenylalanine, the phosphate content (mol/mol hydroxylase) increasing to 0.5 from the basal level (0.17) in 50 s. The half maximal effect of Phenylalanine was obtained at a physiologically relevant concentration (110 μM). The synthetic Phenylalanine hydroxylase cofactor dimethyltetrahydropterin also enhanced the cAMP-stimulated phosporylation of Phenylalanine hydroxylase, presumably by displacing the endogenous cofactor, tetrahydrobiopterin. Phenylalanine was a negative modulator of the phosphorylation of Phenylalanine hydroxylase induced by incubating cells with vasopressin or with the phosphatase inhibitor okadaic acid. The same site on the Phenylalanine hydroxylase was phosphorylated in response to these two agents as in response to elevated cAMP. The available evidence suggested that not only vasopressin, but also okadaic acid, acted by stimulating the multifunctional Ca2+/calmodulin-dependent protein kinase II or a kinase with closely resembling properties.
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modulation by ligands of the phosphorylation state of Phenylalanine 4 Monooxygenase in intact hepatocytes
1990Co-Authors: Anne P Doskeland, Torgeir Flatmark, Olav Karsten Vintermyr, Richard G H Cotton, Stein Ove DoskelandAbstract:1. The rate of phosphorylation of Phenylalanine hydroxylase was studied in isolated rat liver cells, preincubated with 32Pi. The intracellular cAMP-kinase was activated either by a glucagon-stimulated increase of endogenous cAMP or by N6-benzoyl-cAMP. The hydroxylase was isolated by immunoaffinity adsorption on anti-PAH(monoclonal)-Protein A- Sepharose and the 32P incorporation measured by autoradiography.