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Paul F. Fitzpatrick - One of the best experts on this subject based on the ideXlab platform.
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Mutagenesis of an Active-Site Loop in Tryptophan Hydroxylase Dramatically Slows the Formation of an Early Intermediate in Catalysis
2018Co-Authors: Bishnu P. Subedi, Paul F. FitzpatrickAbstract:Solution studies of the aromatic amino acid hydroxylases are consistent with the FeIVO intermediate not forming until both the amino acid and Tetrahydropterin substrates have bound. Structural studies have shown that the positions of active-site loops differs significantly between the free enzyme and the enzyme-amino acid-Tetrahydropterin complex. In tryptophan hydroxylase (TrpH) these mobile loops contain residues 124–134 and 365–371, with a key interaction involving Ile366. The I366N mutation in TrpH results in decreases of 1–2 orders of magnitude in the kcat and kcat/Km values. Single turnover analyses establish that the limiting rate constant for turnover is product release for the wild-type enzyme but is formation of the first detectable intermediate I in catalysis in the mutant enzyme. The mutation does not alter the kinetics of NO binding to the ternary complex nor does it uncouple FeIVO formation from amino acid hydroxylation. The effects on the kcat value of wild-type TrpH of changing viscosity are consistent with rate-limiting product release. While the effect of viscosity on the kcat/KO2 value is small, consistent with reversible oxygen binding, the effects on the kcat/Km values for tryptophan and the Tetrahydropterin are large, with the latter value exceeding the expected limit and varying with the identity of the viscogen. In contrast, the kinetic parameters of I366N TrpH show small changes with viscosity. The results are consistent with binding of the amino acid and pterin substrate to form the ternary complex being directly coupled to closure of loops over the active site and formation of the reactive complex. The mutation destabilizes this initial event
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Kinetic Mechanism and Intrinsic Rate Constants for the Reaction of a Bacterial Phenylalanine Hydroxylase
2016Co-Authors: Bishnu P. Subedi, Paul F. FitzpatrickAbstract:The pterin-dependent aromatic amino acid hydroxylases are non-heme iron enzymes that catalyze the hydroxylation of the aromatic side chain of their respective substrates using an FeIVO intermediate. While the eukaryotic enzymes are homotetramers with complex regulatory properties, bacterial phenylalanine hydroxylases are monomers that lack regulatory domains. As a result, the bacterial enzymes are more tractable for mechanistic studies. Using single turnover methods, the complete kinetic mechanism and intrinsic rate constants for Chromobacterium violaceum phenylalanine hydroxylase have been determined with both tetrahydrobiopterin and 6-methyltetrahyropterin as substrates. In addition the kinetics of formation of the enzyme–pterin complex have been determined with the unreactive 5-deaza, 6-methylTetrahydropterin. For all three pterins, binding of phenylalanine and pterin occurs in random order with binding of the pterin first the preferred pathway. The reaction of the ternary enzyme–phenylalanine–Tetrahydropterin complex can be described by a mechanism involving reversible oxygen binding, formation of an early intermediate preceding formation of the FeIVO, and rate-limiting product release
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evidence for a high spin fe iv species in the catalytic cycle of a bacterial phenylalanine hydroxylase
Biochemistry, 2011Co-Authors: Aram J Panay, Paul F. Fitzpatrick, Michael Lee, Carsten Krebs, Martin J BollingerAbstract:Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses Tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mossbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme−57Fe(II)−phenylalanine−6-methylTetrahydropterin complex with O2 reveal the accumulation of an intermediate at short reaction times (20−100 ms). The Mossbauer parameters of the intermediate (δ = 0.28 mm/s, and |ΔEQ| = 1.26 mm/s) suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a Tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent t...
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regulation of phenylalanine hydroxylase conformational changes upon phenylalanine binding detected by hydrogen deuterium exchange and mass spectrometry
Biochemistry, 2010Co-Authors: Lawrence J Dangott, Paul F. FitzpatrickAbstract:Phenylalanine acts as an allosteric activator of the Tetrahydropterin-dependent enzyme phenylalanine hydroxylase. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into local conformational changes accompanying activation of rat phenylalanine hydroxylase by phenylalanine. Peptides in the regulatory and catalytic domains that lie in the interface between these two domains show large increases in the extent of deuterium incorporation from solvent in the presence of phenylalanine. In contrast, the effects of phenylalanine on the exchange kinetics of a mutant enzyme lacking the regulatory domain are limited to peptides surrounding the binding site for the amino acid substrate. These results support a model in which the N-terminus of the protein acts as an inhibitory peptide, with phenylalanine binding causing a conformational change in the regulatory domain that alters the interaction between the catalytic and regulatory domains.
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direct spectroscopic evidence for a high spin fe iv intermediate in tyrosine hydroxylase
Journal of the American Chemical Society, 2007Co-Authors: Bekir E Eser, Carsten Krebs, Martin J Bollinger, Eric W Barr, Patrick A Frantom, Lana Saleh, Paul F. FitzpatrickAbstract:Tyrosine hydroxylase, a member of the aromatic amino acid hydroxylase family, uses a mononuclear Fe(II) and Tetrahydropterin for hydroxylation of tyrosine to dihydroxyphenylalanine. Rapid-freeze quench Mossbauer spectroscopy has now provided direct evidence for the presence of an Fe(IV) intermediate in the reaction catalyzed by tyrosine hydroxylase. Rapid-quench techniques provide support for the kinetic competence of this species as the hydroxylating intermediate. This is the first direct evidence for a mononuclear Fe(IV) intermediate in an enzymatic aromatic hydroxylation reaction.
Bernd Mayer - One of the best experts on this subject based on the ideXlab platform.
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the pteridine binding site of brain nitric oxide synthase tetrahydrobiopterin binding kinetics specificity and allosteric interaction with the substrate domain
Journal of Biological Chemistry, 1994Co-Authors: Peter Klatt, Ernst R. Werner, Kurt Schmidt, M Schmid, Eva Leopold, Bernd MayerAbstract:Nitric oxide (NO) synthases contain FAD, FMN, heme, and (6R)-5,6,7,8-tetrahydro-L-biopterin as prosthetic groups. We have characterized the pteridine-binding site of purified brain NO synthase, using 3H-labeled (6R)-5,6,7,8-tetrahydro-L-biopterin as radioligand. Association of [3H]tetrahydrobiopterin followed second-order kinetics (kon = 1.3 x 10(6) M-1 min-1), the dissociation reaction was reversible and first-order (koff = 3.2 x 10(-1) min-1), yielding a kinetic KD of 0.25 microM. Binding of the radioligand was competitively antagonized by several pteridine derivatives with the following order of potency (KI): 7,8-dihydro-L-biopterin (2.2 microM), (6S)-5,6,7,8-tetrahydro-L-biopterin (19 microM), (6R,S)-6-methyl-5,6,7,8-Tetrahydropterin (240 microM), and 6,7-dimethyl-5,6,7,8-Tetrahydropterin (> 1 mM). The affinity of NO synthase for tetrahydrobiopterin was increased 6-fold in the presence of 0.1 mM L-arginine (KD = 37 nM), and, conversely, tetrahydrobiopterin enhanced the affinity of the enzyme for 3H-labeled NG-nitro-L-arginine about 2-fold. 7-Nitroindazole, which presumably binds to the heme group of NO synthase, competitively inhibited binding of [3H]tetrahydrobiopterin and [3H]NG-nitro-L-arginine with similar Ki values (0.1 microM). Functional as well as binding studies revealed that 7-nitroindazole was competitive with both L-arginine and tetrahydrobiopterin. Our data indicate that brain NO synthase exhibits a highly specific binding site for (6R)-5,6,7,8-tetrahydro-L-biopterin, which allosterically interacts with the substrate domain and may be located proximal to the prosthetic heme group of NO synthase.
Beat Thony - One of the best experts on this subject based on the ideXlab platform.
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diagnosis of tetrahydrobiopterin deficiency using filter paper blood spots further development of the method and 5 years experience
Journal of Inherited Metabolic Disease, 2011Co-Authors: Thomas Opladen, Beat Thony, Bettina Abu Seda, Anahita Rassi, Georg F HoffmannAbstract:In every newborn with even mild hyperphenyla- laninemia (HPA) tetrahydrobiopterin (BH4) deficiencies need to be excluded as soon as possible. Differential diagnosis is most commonly performed by analysis of urinary neopterin and biopterin. In 2005 a new method for the measurement of neopterin, biopterin and other pterins in dried blood spot (DBS) on filter paper was introduced. In order to evaluate the usefulness of this method as a standard tool for differential diagnosis of HPAs we analyzed neopterin, biopterin, pterin and dihydropteridine reduc- tase activity in DBS from 362 patients with HPA over the period of five years. Age-dependent reference values were established for the HPA population. Sixty-four patients with BH4 deficiency (27 patients with 6- pyruvoyl-Tetrahydropterin synthase deficiency, seven with GTP cyclohydrolase I deficiency, and 30 with dihydrop- teridine reductase) were identified. Reference values for neopterin and biopterin in DBS were calculated for each of the variants. 6-pyruvoyl-Tetrahydropterin synthase and GTP cyclohydrolase I deficiency can be diagnosed by neopterin and biopterin analysis alone, while for diagnosis of dihydropteridine reductase deficiency additional deter- mination of enzyme activity from the same DBS is essential. Regarding test sensitivity, the interpretation of neopterin and biopterin concentration per hemoglobin is more valid than the interpretation of neopterin and biopterin per liter. Percentage of biopterin, of the sum of neopterin and biopterin should always be calculated. In addition, determination of hemoglobin concentration is essential as a measure for efficient extraction of neopterin and biopterin. Although the measurement of neopterin and biopterin in urine is more sensitive due to the higher concentrations present, our data prove the usefulness of their measurement from DBS for the routine diagnosis of BH4 deficiencies. Abbreviations
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regulation of tetrahydrobiopterin biosynthesis by shear stress
Circulation Research, 2007Co-Authors: Julian D Widder, Wei Chen, Sergey Dikalov, Kazuyuki Hatakeyama, Beat Thony, David G HarrisonAbstract:An essential cofactor for the endothelial NO synthase is tetrahydrobiopterin (H4B). In the present study, we show that in human endothelial cells, laminar shear stress dramatically increases H4B levels and enzymatic activity of GTP cyclohydrolase (GTPCH)-1, the first step of H4B biosynthesis. In contrast, protein levels of GTPCH-1 were not affected by shear. Shear did not change protein expression or activity of the downstream enzymes 6-pyruvoyl-Tetrahydropterin synthase and sepiapterin reductase and decreased protein levels of the salvage enzyme dihydrofolate reductase. Oscillatory shear only modestly affected H4B levels and GPTCH-1 activity. We also demonstrate that laminar, but not oscillatory shear stress, stimulates phosphorylation of GTPCH-1 on serine 81 and that this is mediated by the α prime (α′) subunit of casein kinase 2. The increase in H4B caused by shear is essential in allowing proper function of endothelial NO synthase because GPTCH-1 blockade with 2,4-diamino-6-hydroxypyrimidine during sh...
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tetrahydrobiopterin biosynthesis regeneration and functions
Biochemical Journal, 2000Co-Authors: Beat Thony, Gunter Auerbach, Nenad BlauAbstract:Tetrahydrobiopterin (BH % ) cofactor is essential for various processes, and is present in probably every cell or tissue of higher organisms. BH % is required for various enzyme activities, and for less defined functions at the cellular level. The pathway for the de noao biosynthesis of BH % from GTP involves GTP cyclohydrolase I, 6-pyruvoyl-Tetrahydropterin synthase and sepiapterin reductase. Cofactor regeneration requires pterin-4a-carbinolamine dehydratase and dihydropteridine reductase. Based on gene cloning, recombinant expression, mutagenesis studies, structural analysis of crystals and NMR studies, reaction mechanisms for the biosynthetic and recycling enzymes were proposed. With regard to the regulation of cofactor biosynthesis, the major controlling point is GTP cyclohydrolase I, the expression of which may be under the control of cytokine induction. In the liver at least, activity is inhibited by BH % , but stimulated by phenylalanine through the GTP cyclohydrolase I feedback regulatory protein. The enzymes that depend on BH % are the phenylalanine, tyrosine and tryptophan hydroxylases, the latter
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hyperphenylalaninemia due to defects in tetrahydrobiopterin metabolism molecular characterization of mutations in 6 pyruvoyl Tetrahydropterin synthase
American Journal of Human Genetics, 1994Co-Authors: Beat Thony, Walter Leimbacher, Nenad Blau, A Harvie, Claus W HeizmannAbstract:A variant type of hyperphenylalaninemia is caused by a deficiency of tetrahydrobiopterin (BH4), the obligatory cofactor for phenylalanine hydroxylase. The most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-Tetrahydropterin synthase (PTPS) activity, the second enzyme in the biosynthetic pathway for BH4. The human liver cDNA for PTPS was previously isolated, and the recombinant protein was found to be active when expressed in Escherichia coli. We now have investigated two patients for their molecular nature of this autosomal recessive disorder. Both patients were diagnosed as PTPS deficient, one with the central and one with the peripheral form, on the basis of an elevated serum phenylalanine concentration concomitant with lowered levels of urinary biopterin and PTPS activity in erythrocytes. Molecular analysis was performed on the patients' cultured primary skin fibroblasts. PTPS activities were found in vitro to be reduced to background activity. Direct cDNA sequence analysis using reverse transcriptase-PCR technology showed for the patient with the central from a homozygous G-to-A transition at codon 25, causing the replacement of an arginine by glutamine (R25Q). Expression of this mutant allele in E. coli revealed 14% activity when compared with the wild-type enzyme. The patient with the peripheral form exhibited compound heterozygosity, having on one allele a C-to-T transition resulting in the substitution of arginine 16 for cysteine (R16C) in the enzyme and having on the second allele a 14-bp deletion (delta 14bp), leading to a frameshift at lysine 120 and a premature stop codon (K120-->Stop). Heterologous expression of the enzyme with the single-amino-acid exchange R16C revealed only 7% enzyme activity, whereas expression of the deletion allele delta 14bp exhibited no detectable activity. All three mutations, R25Q, R16C, and K120-->Stop, affect evolutionarily conserved residues in PTPS, result in reduced enzymatic activity when reconstituted in E. coli, and are thus believed to be the molecular cause for the BH4 deficiency. This is the first report describing mutations in PTPS that lead to BH4 deficiency.
Peter Klatt - One of the best experts on this subject based on the ideXlab platform.
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the pteridine binding site of brain nitric oxide synthase tetrahydrobiopterin binding kinetics specificity and allosteric interaction with the substrate domain
Journal of Biological Chemistry, 1994Co-Authors: Peter Klatt, Ernst R. Werner, Kurt Schmidt, M Schmid, Eva Leopold, Bernd MayerAbstract:Nitric oxide (NO) synthases contain FAD, FMN, heme, and (6R)-5,6,7,8-tetrahydro-L-biopterin as prosthetic groups. We have characterized the pteridine-binding site of purified brain NO synthase, using 3H-labeled (6R)-5,6,7,8-tetrahydro-L-biopterin as radioligand. Association of [3H]tetrahydrobiopterin followed second-order kinetics (kon = 1.3 x 10(6) M-1 min-1), the dissociation reaction was reversible and first-order (koff = 3.2 x 10(-1) min-1), yielding a kinetic KD of 0.25 microM. Binding of the radioligand was competitively antagonized by several pteridine derivatives with the following order of potency (KI): 7,8-dihydro-L-biopterin (2.2 microM), (6S)-5,6,7,8-tetrahydro-L-biopterin (19 microM), (6R,S)-6-methyl-5,6,7,8-Tetrahydropterin (240 microM), and 6,7-dimethyl-5,6,7,8-Tetrahydropterin (> 1 mM). The affinity of NO synthase for tetrahydrobiopterin was increased 6-fold in the presence of 0.1 mM L-arginine (KD = 37 nM), and, conversely, tetrahydrobiopterin enhanced the affinity of the enzyme for 3H-labeled NG-nitro-L-arginine about 2-fold. 7-Nitroindazole, which presumably binds to the heme group of NO synthase, competitively inhibited binding of [3H]tetrahydrobiopterin and [3H]NG-nitro-L-arginine with similar Ki values (0.1 microM). Functional as well as binding studies revealed that 7-nitroindazole was competitive with both L-arginine and tetrahydrobiopterin. Our data indicate that brain NO synthase exhibits a highly specific binding site for (6R)-5,6,7,8-tetrahydro-L-biopterin, which allosterically interacts with the substrate domain and may be located proximal to the prosthetic heme group of NO synthase.
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The pteridine binding site of brain nitric oxide synthase. Tetrahydrobiopterin binding kinetics, specificity, and allosteric interaction with the substrate domain
1994Co-Authors: Peter Klatt, Ernst R. Werner, Kurt Schmidt, M Schmid, Eva Leopold, Bernd MayeroAbstract:and (6R)-5,6,7,84etrahydro-~-biopterin as prosthetic groups. We have characterized the pteridine-binding site of purified brain NO synthase, using 'H-labeled (6R)-5,6,7,8-tetrahydro-~-biopterin as radioligand. Asso-ciation of ['Hltetrahydrobiopterin followed second-or-der kinetics (ken = 1.3 x 10 ' M- ~ m i d) , the dissociation reaction was reversible and first-order (kOE = 3.2 x 10" min"), yielding a kinetic KD of 0.25 p. Binding of the radioligand was competitively antagonized by several pteridine derivatives with the following order of po-tency (KI): 7,8-dihydro-~-biopterin (2.2 p ~ ) , (65)-5,6,7,8-tetrahydro-L-biopterin (19 p), (6R,S)-6-methyl-5,6,7,8-Tetrahydropterin (240 p), and 6,7-dimethyl-5,6,7,8-Tetrahydropterin (>1 m). The affinity of NO synthase for tetrahydrobiopterin was increased 6-fold in the pres-ence of 0.1 m ~ L-arginine (K, = 37 m), and, conversely, tetrahydrobiopterin enhanced the affinity of the en-zyme for 'H-labele
Carsten Krebs - One of the best experts on this subject based on the ideXlab platform.
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evidence for a high spin fe iv species in the catalytic cycle of a bacterial phenylalanine hydroxylase
Biochemistry, 2011Co-Authors: Aram J Panay, Paul F. Fitzpatrick, Michael Lee, Carsten Krebs, Martin J BollingerAbstract:Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses Tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mossbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme−57Fe(II)−phenylalanine−6-methylTetrahydropterin complex with O2 reveal the accumulation of an intermediate at short reaction times (20−100 ms). The Mossbauer parameters of the intermediate (δ = 0.28 mm/s, and |ΔEQ| = 1.26 mm/s) suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a Tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent t...
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direct spectroscopic evidence for a high spin fe iv intermediate in tyrosine hydroxylase
Journal of the American Chemical Society, 2007Co-Authors: Bekir E Eser, Carsten Krebs, Martin J Bollinger, Eric W Barr, Patrick A Frantom, Lana Saleh, Paul F. FitzpatrickAbstract:Tyrosine hydroxylase, a member of the aromatic amino acid hydroxylase family, uses a mononuclear Fe(II) and Tetrahydropterin for hydroxylation of tyrosine to dihydroxyphenylalanine. Rapid-freeze quench Mossbauer spectroscopy has now provided direct evidence for the presence of an Fe(IV) intermediate in the reaction catalyzed by tyrosine hydroxylase. Rapid-quench techniques provide support for the kinetic competence of this species as the hydroxylating intermediate. This is the first direct evidence for a mononuclear Fe(IV) intermediate in an enzymatic aromatic hydroxylation reaction.