guanosine-triphosphate and sapropterin

Topics: Alcohol Oxidoreductases; Amino Acid Metabolism, Inborn Errors; Biopterins; Diagnosis, Differential; Female; Guanosine Triphosphate; Homovanillic Acid; Humans; Hydroxyindoleacetic Acid; Phenylalanine; Phosphorus-Oxygen Lyases; Pregnancy; Prenatal Diagnosis

The apolipoprotein A-I mimetic peptide D-4F, among its anti-atherosclerotic effects, improves vasodilation through mechanisms not fully elucidated yet.. Low-density lipoprotein (LDL) receptor null (LDLr. Unexpectedly, eNOS phosphorylation, eNOS-HSP90 association, and O. Hypercholesterolemia enhanced uncoupled eNOS activity by decreasing GCH-1 concentration, thereby reducing BH4 levels. D-4F reduced uncoupled eNOS activity by increasing BH4 levels through GCH-1 expression and decreasing eNOS phosphorylation and eNOS-HSP90 association. Our findings elucidate a novel mechanism by which hypercholesterolemia induces atherosclerosis and D-4F inhibits it, providing a potential therapeutic approach.

Topics: Animals; Apolipoprotein A-I; Atherosclerosis; Biopterins; Endothelial Cells; Endothelium, Vascular; GTP Cyclohydrolase; Guanosine Triphosphate; Mice; Nitric Oxide; Nitric Oxide Synthase Type III; Peptides; Superoxides

Parkinson disease (PD) is a multifactorial disease resulting in preferential death of the dopaminergic neurons in the substantia nigra. Studies of PD-linked genes and toxin-induced models of PD have implicated mitochondrial dysfunction, oxidative stress, and the misfolding and aggregation of α-synuclein (α-syn) as key factors in disease initiation and progression. Many of these features of PD may be modeled in cells or animal models using the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)). Reducing oxidative stress and nitric oxide synthase (NOS) activity has been shown to be protective in cell or animal models of MPP(+) toxicity. We have previously demonstrated that siRNA-mediated knockdown of α-syn lowers the activity of both dopamine transporter and NOS activity and protects dopaminergic neuron-like cells from MPP(+) toxicity. Here, we demonstrate that α-syn knockdown and modulators of oxidative stress/NOS activation protect cells from MPP(+)-induced toxicity via postmitochondrial mechanisms rather than by a rescue of the decrease in mitochondrial oxidative phosphorylation caused by MPP(+) exposure. We demonstrate that MPP(+) significantly decreases the synthesis of the antioxidant and obligate cofactor of NOS and TH tetrahydrobiopterin (BH4) through decreased cellular GTP/ATP levels. Furthermore, we demonstrate that RNAi knockdown of α-syn results in a nearly twofold increase in GTP cyclohydrolase I activity and a concomitant increase in basal BH4 levels. Together, these results demonstrate that both mitochondrial activity and α-syn play roles in modulating cellular BH4 levels.

Topics: 1-Methyl-4-phenylpyridinium; Adenosine Triphosphate; alpha-Synuclein; Biopterins; Cell Line, Tumor; Dopaminergic Neurons; Gene Expression Regulation; GTP Cyclohydrolase; Guanosine Triphosphate; Humans; Mitochondria; Models, Biological; Oxidative Phosphorylation; Parkinson Disease; RNA, Small Interfering

GTP cyclohydrolases generate the first committed intermediates for the biosynthesis of certain vitamins/cofactors (folic acid, riboflavin, deazaflavin, and tetrahydrobiopterin), deazapurine antibiotics, some t-RNA bases (queuosine, archaeosine), and the phytotoxin, toxoflavin. They depend on divalent cations for hydrolytic opening of the imidazole ring of the substrate, guanosine triphosphate (GTP). Surprisingly, the ring opening reaction is not the rate-limiting step for GTP cyclohydrolases I and II whose mechanism have been studied in some detail. GTP cyclohydrolase I, Ib, and II are potential targets for novel anti-infectives. Genetic factors modulating the activity of human GTP cyclohydrolase are highly pleiotropic, since the signal transponders whose biosyntheses require their participation (nitric oxide, catecholamines) impact a very wide range of physiological phenomena. Recent studies suggest that human GTP cyclohydrolase may become an oncology target.

Topics: Anti-Bacterial Agents; Biopterins; Cations, Divalent; Escherichia coli; Folic Acid; GTP Cyclohydrolase; Guanosine Triphosphate; Humans; Kinetics; Riboflavin

Vascular smooth muscle cell (VSMC) proliferation is pivotal in the progression of hypertension, atherosclerosis, and restenosis. Resveratrol is a grape polyphenol that is implicated as an important contributor to red wine's vascular protective effects. Its antimitogenic action on VSMC is attributed to an array of pleiotropic effects, including modulation of the estrogen receptor (ER). To elucidate the mechanisms underlying resveratrol-mediated ER modulation and its inhibition of VSMC proliferation, we treated VSMC with resveratrol with or without the ER antagonist ICI 182,780 and measured cell proliferation and nitric oxide (NO) production. Resveratrol dose-dependently decreased VSMC DNA synthesis, with a half maximal inhibitory concentration (IC50) of 3.73+/-0.57 microM, and dramatically slowed cell growth, but did not induce VSMC apoptosis. Resveratrol-mediated decrease in proliferation was reversed by cotreatment with ICI 182,780, and resveratrol effectively competed with 17beta-estradiol for binding to the ER, exhibiting an IC50 of 8.92+/-0.14 microM. Resveratrol induced a sustained increase in ER-dependent NO production. Further, resveratrol-mediated decrease in VSMC proliferation was blunted by cotreatment with the general nitric oxide synthase (NOS) inhibitor N5-(1-Iminomethyl)-L-ornithine, dihydrochloride or with the inducible NOS (iNOS)-selective inhibitor S,S'-1,4-phenylene-bis (1,2-ethanediyl)bis-isothiourea, dihydrobromide, but not with the neuronal NOS-selective inhibitor 7-nitroindazole. Though resveratrol did not alter iNOS protein levels, it dose-dependently increased levels of iNOS activity, of the iNOS cofactor tetrahydrobiopterin (BH4), and of guanosine triphosphate cyclohydrolase I protein, the rate-limiting enzyme in BH4 biosynthesis. In addition, all of these effects were abolished by cotreatment with ICI 182,780. Thus, the antimitogenic effects of resveratrol on VSMC may be mediated by an ER-induced increase in iNOS activity.

Topics: Animals; Antioxidants; Aorta; Apoptosis; Biopterins; Cell Cycle; Cell Proliferation; DNA; Dose-Response Relationship, Drug; Female; Guanosine Triphosphate; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Receptors, Estrogen; Resveratrol; Stilbenes

Tetrahydrobiopterin (BH4) deficiency is reported to uncouple the enzymatic activity of endothelial nitric oxide synthase in diabetes mellitus. The mechanism by which diabetes actually leads to BH4 deficiency remains elusive. Here, we demonstrate that diabetes reduced BH4 by increasing 26S proteasome-dependent degradation of guanosine 5'-triphosphate cyclohydrolase I (GTPCH), a rate-limiting enzyme in the synthesis of BH4, in parallel with increased formation of both superoxide and peroxynitrite (ONOO-).. Exposure of human umbilical vein endothelial cells to high glucose concentrations (30 mmol/L D-glucose) but not to high osmotic conditions (25 mmol/L L-glucose plus 5 mmol/L D-glucose) significantly lowered the levels of both GTPCH protein and BH4. In addition, high glucose increased both the 26S proteasome activity and the ubiquitination of GTPCH. Inhibition of the 26S proteasome with either MG132 or PR-11 prevented the high glucose-triggered reduction of GTPCH and BH4. Exposure of human umbilical vein endothelial cells to exogenous ONOO- increased proteasome activity and 3-nitrotyrosine in 26S proteasome. Furthermore, adenoviral overexpression of superoxide dismutase and inhibition of endothelial nitric oxide synthase with N(G)-nitro-L-arginine methyl ester significantly attenuated the high glucose-induced activation of 26S proteasome and the reduction of GTPCH. Finally, administration of MG132 or a superoxide dismutase mimetic, tempol, reversed the diabetes mellitus-induced reduction of GTPCH and BH4 and endothelial dysfunction in streptozotocin-induced diabetes mellitus.. We conclude that diabetes mellitus triggers BH4 deficiency by increasing proteasome-dependent degradation of GTPCH.

Topics: Animals; Antioxidants; Aorta; Biopterins; Cells, Cultured; Cyclic N-Oxides; Cysteine Proteinase Inhibitors; Diabetes Mellitus, Experimental; Endothelial Cells; Glucose; GTP Cyclohydrolase; Guanosine Triphosphate; Leupeptins; Mice; Mice, Inbred C57BL; Nitrogen; Organ Culture Techniques; Peroxynitrous Acid; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Reactive Oxygen Species; Spin Labels; Tyrosine; Ubiquitin; Umbilical Veins

The yeast 2-hybrid system was used to identify protein domains involved in the oligomerization of human guanosine 5'-triphosphate (GTP) Cyclohydrolase I (GCH1) and the interaction of GCH1 with its regulatory partner, GCH1 feedback regulatory protein (GFRP). When interpreted within the structural framework derived from crystallography, our results indicate that the GCH1 N-terminal alpha-helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C-terminal alpha-helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N-terminal alpha-helix in the interaction of GCH1 and GFRP was revealed. To discover novel GCH1 protein binding partners, we used the yeast 2-hybrid system to screen a human brain library with GCH1 N-terminal amino acids 1-96 as prey. This protruding extension of GCH1 contains two canonical Type-I Src homology-3 (SH3) ligand domains located within amino acids 1-42. Our screen yielded seven unique clones that were subsequently shown to require amino acids 1-42 for binding to GCH1. The interaction of one of these clones, Activator of Heat Shock 90 kDa Protein (Aha1), with GCH1 was validated by glutathione-s-transferase (GST) pull-down assay. Although the physiological relevance of the Aha1-GCH1 interaction requires further study, Aha1 may recruit GCH1 into the endothelial nitric oxide synthase/heat shock protein (eNOS/Hsp90) complex to support changes in endothelial nitric oxide production through the local synthesis of BH4.

Topics: Amino Acid Sequence; Binding Sites; Biopterins; Chaperonins; Crystallography, X-Ray; Endothelium, Vascular; Enzyme Activation; Gene Library; GTP Cyclohydrolase; Guanosine Triphosphate; HSP90 Heat-Shock Proteins; Humans; Intracellular Signaling Peptides and Proteins; Molecular Sequence Data; Nitric Oxide; Nitric Oxide Synthase Type III; Polymers; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Two-Hybrid System Techniques

GTP cyclohydrolase I (GCH) is the rate-limiting enzyme for the synthesis of tetrahydrobiopterin and its activity is important in the regulation of monoamine neurotransmitters such as dopamine, norepinephrine and serotonin. We have studied the action of divalent cations on the enzyme activity of purified recombinant human GCH expressed in Escherichia coli. First, we showed that the enzyme activity is dependent on the concentration of Mg-free GTP. Inhibition of the enzyme activity by Mg2+, as well as by Mn2+, Co2+ or Zn2+, was due to the reduction of the availability of metal-free GTP substrate for the enzyme, when a divalent cation was present at a relatively high concentration with respect to GTP. We next examined the requirement of Zn2+ for enzyme activity by the use of a protein refolding assay, because the recombinant enzyme contained approximately one zinc atom per subunit of the decameric protein. Only when Zn2+ was present was the activity of the denatured enzyme effectively recovered by incubation with a chaperone protein. These are the first data demonstrating that GCH recognizes Mg-free GTP and requires Zn2+ for its catalytic activity. We suggest that the cellular concentration of divalent cations can modulate GCH activity, and thus tetrahydrobiopterin biosynthesis as well.

Topics: Biopterins; Cations, Divalent; Cobalt; Escherichia coli; GTP Cyclohydrolase; Guanosine Triphosphate; Humans; Iron; Magnesium; Manganese; Recombinant Proteins; Substrate Specificity

The activity of GTP cyclohydrolase I is inhibited by (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and stimulated by phenylalanine through complex formation with GTP cyclohydrolase I feedback regulatory protein (GFRP). Gel filtration experiments as well as enzyme activity measurements showed that the number of subunits of GFRP in both the inhibitory and stimulatory complexes is equal to that of GTP cyclohydrolase I. Because GFRP is a pentamer and GTP cyclohydrolase I was shown here by cross-linking experiments to be a decamer, the results indicate that two molecules of a pentameric GFRP associate with one molecule of GTP cyclohydrolase I. Gel filtration analysis suggested that the complex has a radius of gyration similar to that of the enzyme itself. These observations support our model that one molecule of GFRP binds to each of the two outer faces of the torus-shaped GTP cyclohydrolase I. For formation of the inhibitory protein complex, both BH4 and GTP were required; the median effective concentrations of BH4 and GTP were 2 and 26 microM, respectively. BH4 was the most potent of biopterins with different oxidative states. Among GTP analogues, dGTP as well as guanosine 5'-O-(3'-thiotriphosphate) exhibited similar inducibility compared with GTP, whereas other nucleotide triphosphates had no effect. On the other hand, phenylalanine alone was enough for formation of the stimulatory protein complex, and positive cooperativity was found for the phenylalanine-induced protein complex formation. Phenylalanine was the most potent of the aromatic amino acids.

Topics: Animals; Biopterins; Chromatography, Gel; Cross-Linking Reagents; Dimethyl Suberimidate; GTP Cyclohydrolase; Guanosine Triphosphate; Intracellular Signaling Peptides and Proteins; Phenylalanine; Protein Binding; Protein Conformation; Proteins; Rats; Recombinant Proteins

Hepatic phenylalanine hydroxylase is reported to be more abundant in experimentally-diabetic rats; whereas livers of animals fed a high protein diet, where gluconeogenesis also prevails, have normal amounts of this enzyme. In this study, in addition to seeking an explanation for this effect of experimental diabetes, we also examined the effects of providing alternative dietary gluconeogenic substrates. In rats fed a diet composed of 40% (w/w) glycerol, the specific activities of hepatic phenylalanine hydroxylase are decreased to about 60% of control values. There is no effect on the apparent state of phosphorylation of the enzyme. However, studies on the incorporation of radiolabelled leucine into liver phenylalanine hydroxylase suggested that there was a decreased rate of synthesis. Similarly, animals fed a diet containing 85% (w/w) fructose also have diminished phenylalanine hydroxylase activities. Under all of the above circumstances and also in streptozotocin-induced diabetic animals, alterations in the concentrations of the hydroxylase cofactor, tetrahydrobiopterin and of GTP closely correlate with the effects on the enzyme activities. They are elevated in livers of diabetic animals and significantly diminished in livers of rats fed diets rich in glycerol or fructose. These observations suggest that in adult rat both liver tetrahydrobiopterin concentrations and the expression of hepatic phenylalanine hydroxylase are regulated by GTP [210].

Topics: Animals; Biopterins; Diabetes Mellitus, Experimental; Dietary Sucrose; Fructose; Glycerol; Guanosine Triphosphate; Kinetics; Liver; Male; Nucleotides; Phenylalanine Hydroxylase; Rats; Rats, Sprague-Dawley

Topics: Animals; Biopterins; Blotting, Western; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Guanosine Triphosphate; Liver; Male; Phenylalanine Hydroxylase; Rats; Rats, Sprague-Dawley

Topics: Alcohol Oxidoreductases; Animals; Biopterins; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Escherichia coli; GTP Cyclohydrolase; Guanosine Triphosphate; Kinetics; Phenylalanine Hydroxylase; Phosphorus-Oxygen Lyases; Rats; Stereoisomerism

6-(D-threo-1',2'-Dihydroxypropylpterin (dictyopterin) has been identified in extracts of growing Dictyostelium dicoideum cells [Klein, Thiery and Tatischeff (1990) Eur. J. Biochem. 187, 665-669]. We demonstrate that it originates from GTP by de novo biosynthesis and that the first committed step is catalysed by GTP cyclohydrolase I, yielding dihydroneopterin triphosphate [neopterin is 6-(D-erythro-1',2',3'-trihydroxypropyl) pterin]. The GTP cyclohydrolase I activity is found in the cytosolic fraction and in a membrane-associated form. The level of a 0.9 kb mRNA coding for GTP cyclohydrolase I decreases to about 10% of its initial value within 2 h after Dictyostelium cells start development induced by starvation. In the cytosolic fraction, the specific activities of GTP cyclohydrolase I, as well as the concentrations of (6R/S)-5,6,7,8-tetrahydrodictyopterin (H4dictyopterin), follow this decline of the mRNA level. In the particulate fraction, however, the specific activities of GTP cyclohydrolase I and, in consequence, H4dictyopterin synthesis, transiently increase and reach a maximum after 4-5 h of development. The time-course of H4dictyopterin concentrations in the starvation medium closely correlates with its production in the membrane fraction. The activity of membrane-associated GTP cyclohydrolase I can be increased by pre-incubation of the cell lysate with guanosine 5'-[gamma-thio]triphosphate and Mg2+. This GTP analogue does not serve as a substrate and has no direct effect on the enzyme activity, indicating that a G-protein-linked signalling pathway is involved in the regulation of GTP cyclohydrolase I activity and thus in H4dictyopterin production during early development of D. discoideum.

Topics: Alcohol Oxidoreductases; Amino Acid Sequence; Animals; Biopterins; Blotting, Northern; Chromatography, High Pressure Liquid; Dictyostelium; Enzyme Activation; Gene Expression; GTP Cyclohydrolase; GTP-Binding Proteins; Guanosine Triphosphate; Molecular Sequence Data; Molecular Structure; Phosphorus-Oxygen Lyases; Pterins; RNA, Messenger; Signal Transduction

Tetrahydrobiopterin serves as the cofactor for enzymes involved in neurotransmitter biosynthesis and as regulatory factor in immune cell proliferation and the biosynthesis of melanin. The biosynthetic pathway to tetrahydrobiopterin consists of three steps starting from GTP. The initial reaction is catalyzed by GTP cyclohdrolase I (GTP-CH-I) and involves the chemically complex transformation of the purine into the pterin ring system.. The crystal structure of the Escherichia coli GTP-CH-I was solved by single isomorphous replacement and molecular averaging at 3.0 A resolution. The functional enzyme is a homodecameric complex with D5 symmetry, forming a torus with dimensions 65 A x 100 A. The pentameric subunits are constructed via an unprecedented cyclic arrangement of the four-stranded antiparallel beta-sheets of the five monomers to form a 20-stranded antiparallel beta-barrel of 35 A diameter. Two pentamers are tightly associated by intercalation of two antiparallel helix pairs positioned close to the subunit N termini. The C-terminal domain of the GTP-CH-I monomer is topologically identical to a subunit of the homohexameric 6-pyruvoyl tetrahydropterin synthase, the enzyme catalyzing the second step in tetrahydrobiopterin biosynthesis.. The active site of GTP-CH-I is located at the interface of three subunits. It represents a novel GTP-binding site, distinct from the one found in G proteins, with a catalytic apparatus that suggest involvement of histidines and, possibly, a cystine in the unusual reaction mechanism. Despite the lack of significant sequence homology between GTP-CH-I and 6-pyruvoyl tetrahydropterin synthase, the two proteins, which catalyze consecutive steps in tetrahydrobiopterin biosynthesis, share a common subunit fold and oligomerization mode. In addition, the active centres have an identical acceptor site for the 2-amino-4-oxo pyrimidine moiety of their substrates which suggests an evolutionarily conserved protein fold designed for pterin biosynthesis.

Topics: Alcohol Oxidoreductases; Bacterial Proteins; Binding Sites; Biopterins; Catalysis; Crystallography, X-Ray; Escherichia coli; GTP Cyclohydrolase; Guanosine Triphosphate; Models, Molecular; Neopterin; Phosphorus-Oxygen Lyases; Protein Conformation; Pteridines

Treatment of mesangial cells with recombinant human interleukin 1 beta (IL-1 beta) triggers the expression of a macrophage-type of nitric oxide (NO) synthase and the subsequent increase of cellular concentration of cGMP and nitrite production. Tetrahydrobiopterin (BH4) is an essential cofactor for NO synthase, and in the present study we investigated its impact on inducible NO synthesis in mesangial cells. Inhibition of GTP-cyclohydrolase I, the rate-limiting enzyme for BH4 synthesis, with 2,4-diamino-6-hydroxy-pyrimidine (DAHP) potently suppresses IL-1 beta-induced nitrite production and elevation of cellular cGMP levels. This inhibitory effect of DAHP is reversed by sepiapterin, which provides BH4 via the pterin salvage pathway. Most importantly, sepiapterin dose-dependently augments IL-1 beta-stimulated NO synthesis, indicating that the availability of BH4 limits the production of NO in cytokine-induced mesangial cells. N-acetylserotonin, an inhibitor of the BH4 synthetic enzyme sepiapterin reductase, completely abolishes IL-1 beta-stimulated nitrite production, whereas methotrexate, which inhibits the pterin salvage pathway, displays only a moderate inhibitory effect, thus suggesting that mesangial cells predominantly synthesize BH4 by de novo synthesis from GTP. In conclusion, these data demonstrate that BH4 synthesis is an absolute requirement for, and limits IL-1 beta induction of NO synthesis in mesangial cells. Inhibition of BH4 synthesis may provide new therapeutic approaches to the treatment of pathological conditions involving increased NO formation.

Topics: Amino Acid Oxidoreductases; Animals; Biopterins; Cells, Cultured; Dose-Response Relationship, Drug; Glomerular Mesangium; GTP Cyclohydrolase; Guanosine Triphosphate; Hypoxanthines; Interleukin-1; Nitric Oxide; Nitric Oxide Synthase; Pteridines; Pterins; Rats; Recombinant Proteins

Extracts from rat corpus striatum, or striatal proteins resolved by chromatography on DE-52, were tested for protein phosphatase activity using tyrosine hydroxylase, phosphorylated by cAMP-dependent protein kinase, as substrate. The predominant dephosphorylating activity was independent of divalent cations and was inhibited by low concentrations (100 nM) of okadaic acid, defining the phosphatase as type 2A. Phosphatase type 2C (Mg2+ and Mn2+ stimulated) was evident in the presence of okadaic acid but at a level of approximately 10% of type 2A activity. Phosphatase 2B (Ca2+ and calmodulin dependent) mediated dephosphorylation of tyrosine hydroxylase was not apparent. The dephosphorylation of [32P]-tyrosine hydroxylase was not modulated by tetrahydrobiopterin, ATP, or GTP. These results indicate that tyrosine hydroxylase which has been phosphorylated by cAMP dependent protein kinase is dephosphorylated predominantly by phosphatase type 2A in brain, and the activity of this phosphatase is not modulated by pteridines or nucleotides.

Topics: Adenosine Triphosphate; Animals; Biopterins; Brain; Chromatography, Ion Exchange; Cyclic AMP-Dependent Protein Kinases; Dopamine; Ethers, Cyclic; Guanosine Triphosphate; Isoenzymes; Neostriatum; Okadaic Acid; PC12 Cells; Phosphoprotein Phosphatases; Phosphorylation; Rats; Tyrosine 3-Monooxygenase

Topics: Alcohol Oxidoreductases; Animals; Biopterins; Cell Line; Dihydropteridine Reductase; GTP Cyclohydrolase; Guanosine Triphosphate; Kinetics; Mice; Mycophenolic Acid; Neuroblastoma; Phosphorus-Oxygen Lyases; Tumor Cells, Cultured

An enzymatic method for preparing 6R-[U-14C]-tetrahydrobiopterin from [U-14C]GTP is presented. This method utilizes purified preparations of three biosynthetic enzymes for 6R-tetrahydrobiopterin, i.e., Escherichia coli GTP cyclohydrolase I, rat 6-pyruvoyl-tetrahydropterin synthase, and rat sepiapterin reductase. Based on the catalytic properties of these enzymes, the reaction conditions were optimized to complete the entire conversion reaction from GTP to 6R-tetrahydrobiopterin in a single reaction solution without the need to isolate unstable intermediates after each enzymatic reaction. The reaction conditions thereby established yielded [U-14C]biopterin in an amount equivalent to 75%, on a molar basis, of the initial amount of [U-14C]GTP. The product was subsequently isolated by high-performance liquid chromatography. The method produced labeled 6R-tetrahydrobiopterin with a specific activity of 450 Ci/mol and an overall yield of more than 60%.

Topics: Alcohol Oxidoreductases; Animals; Biopterins; Carbon Radioisotopes; Escherichia coli; GTP Cyclohydrolase; Guanosine Triphosphate; Hydrogen-Ion Concentration; Isotope Labeling; Kinetics; Phenylalanine Hydroxylase; Phosphorus-Oxygen Lyases; Rats; Time Factors

GTP cyclohydrolase I exhibits a positive homotropic cooperative binding to GTP, which raises the possibility of a role for GTP in regulating the enzyme reaction (Hatakeyama, K., Harada, T., Suzuki, S., Watanabe, Y., and Kagamiyama, H. (1989) J. Biol. Chem. 264, 21660-21664). We examined whether or not the intracellular GTP level is within the range of affecting GTP cyclohydrolase I activity, using PC-12 rat pheochromocytoma and IMR-32 human neuroblastoma cells. Since GTP cyclohydrolase I was the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in these cell lines, the intracellular activities of this enzyme were reflected in the tetrahydrobiopterin contents. We found that the addition of guanine or guanosine increased GTP but not tetrahydrobiopterin in these cells. On the other hand, three IMP dehydrogenase inhibitors, tiazofurin, 2-amino-1,3,4-thiadiazole, and mycophenolic acid, decreased both GTP and tetrahydrobiopterin in a parallel and dose-dependent manner, and these effects were reversed by the simultaneous addition of guanine or guanosine. There was no evidence suggesting that these inhibitors inhibited other enzymes involved in the biosynthesis and regeneration of tetrahydrobiopterin. Comparing intracellular activities of GTP cyclohydrolase I in the inhibitor-treated cells with its substrate-velocity curve, we estimated that the intracellular concentration of free GTP is 150 microM at which point the activity of GTP cyclohydrolase I is elicited at its maximum velocity. Below this GTP concentration, GTP cyclohydrolase I activity is rapidly decreased. Therefore GTP can be a regulator for tetrahydrobiopterin biosynthesis.

Topics: Animals; Antineoplastic Agents; Biopterins; GTP Cyclohydrolase; Guanine; Guanosine; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Kinetics; Mycophenolic Acid; Neuroblastoma; PC12 Cells; Ribavirin; Thiadiazoles; Tumor Cells, Cultured

The kinetic and regulatory properties of GTP cyclohydrolase I were investigated using an improved enzyme assay and direct determination of the product, dihydroneopterin triphosphate. The enzyme was purified from Escherichia coli to absolute homogeneity as demonstrated by N-terminal sequencing of up to 50 amino acid residues. A 30-residue internal fragment showed 42% similarity with rat liver GTP cyclohydrolase I. The enzyme did not obey Michaelis-Menten kinetics or show a sigmoid reaction curve. The substrate saturation kinetics were found to be slow with low response to minor changes in GTP concentrations. GTP cyclohydrolase I has a relatively high apparent Km. The values are slightly different for enzyme purified by GTP-agarose (100 microM) and UTP-agarose (110 microM). Low turnover numbers of 12/min and 19/min were calculated for the respective enzyme preparations. GTP-cyclohydrolase-I activity was modulated in Vmax by K+, divalent cations, UTP and tetrahydrobiopterin. Divalent cations, such as Mg2+, had an activating effect with an optimum at 8 mM Mg2+. A different catalytic function and formation of a new, unidentified product by GTP cyclohydrolase I was observed in the presence of Ca2+. In the presence of 1 mM EDTA and Mg2+, GTP-cyclohydrolase-I activity was strongly inhibited by chelate complexes. UTP proved not to be a competitive inhibitor, but a positive modulator. The inhibition by chelate complexes was totally abolished by UTP. Tetrahydrobiopterin showed an inhibitory effect, with 50% inhibition at 100 microM tetrahydrobiopterin. UTP was able to reduce the inhibition by tetrahydrobiopterin. Using monoclonal antibody 1F11 (related to the GTP-binding site), and monoclonal antibody NS7 (mimicking tetrahydrobiopterin), different binding sites were demonstrated for GTP and tetrahydrobiopterin on each enzyme subunit. Western-blot competition analysis revealed a UTP-binding site different from the binding sites of GTP and tetrahydrobiopterin. Based on the kinetic behaviour and the kind of modulations observed we defined GTP cyclohydrolase I as an M-class allosteric enzyme.

Topics: Allosteric Regulation; Amino Acid Sequence; Binding Sites; Biopterins; Calcium; Cations, Divalent; Chromatography, Affinity; Edetic Acid; Enzyme Activation; Escherichia coli; GTP Cyclohydrolase; Guanosine Triphosphate; Kinetics; Magnesium; Molecular Sequence Data; Peptide Fragments; Sequence Homology, Amino Acid; Uridine Triphosphate

Salmon liver was chosen for the isolation of 6-pyruvoyl tetrahydropterin synthase, one of the enzymes involved in tetrahydrobiopterin biosynthesis. A 9500-fold purification was obtained and the purified enzyme showed two single bands of 16 and 17 kDa on SDS/PAGE. The native enzyme (68 kDa) consists of four subunits and needs free thiol groups for enzymatic activity as was shown by reacting the enzyme with the fluorescent thiol reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide. The enzyme is heat-stable up to 80 degrees C, has an isoelectric point of 6.0-6.3, and a pH optimum at 7.5. The enzyme is Mg2+ -dependent and has a Michaelis constant for its substrate dihydroneopterin triphosphate of 2.2 microM. The turnover number of the purified salmon liver enzyme is about 50 times as high as that of the enzyme purified from human liver. It does not bind to the lectin concanavalin A, indicating that it is free of mannose and glucose residues. Polyclonal antibodies raised against the purified enzyme in Balb/c mice were able to immunoprecipitate enzyme activity. The same polyclonal serum was not able to immunoprecipitate enzyme activity of human liver 6-pyruvoyl tetrahydropterin synthase, nor was any cross-reaction in ELISA tests seen.

Topics: Alcohol Oxidoreductases; Animals; Antibody Formation; Biopterins; Enzyme Activation; Enzyme Stability; Enzyme-Linked Immunosorbent Assay; Guanosine Triphosphate; Hot Temperature; Liver; Maleimides; Peptide Mapping; Phosphorus-Oxygen Lyases; Salmon; Sulfhydryl Compounds

Topics: Biopterins; Brain; GTP Cyclohydrolase; Guanosine Triphosphate; Humans; Parkinson Disease; Radioimmunoassay

Tetrahydrobiopterin, the cofactor for the aromatic amino acid hydroxylases, is synthesized in mammals from GTP via a pathway involving both dihydropterin and tetrahydropterin intermediates. In this work, we have investigated the mechanism of conversion of the product formed from GTP, 7,8-dihydroneopterin triphosphate, into the tetrahydropterin intermediates. Tetrahydrobiopterin can be oxidized under conditions which yield pterin or pterin 6-carboxylate without exchange of the C-6 and C-7 protons. Using these techniques, a gas chromatography/mass spectrometry method was developed to determine that in the biosynthesis of tetrahydrobiopterin de novo, in preparations of bovine adrenal medulla, the C-6 proton of tetrahydrobiopterin is derived from water and not from NADPH. In contrast, the C-6 proton of tetrahydrobiopterin produced from sepiapterin (6-lactoyl-7,8-dihydropterin) comes from NADPH. The results are consistent with evidence for the formation of the first tetrahydropterin intermediate by a tautomerization without any requirement for NADPH.

Topics: Animals; Biopterins; Cattle; Deuterium; Gas Chromatography-Mass Spectrometry; Guanosine Triphosphate; Models, Chemical; NADP; Protons; Pteridines

Mammalian cells and tissues were found to have two pathways for the biosynthesis of tetrahydrobiopterin (BH4): (i) the conversion of GTP to BH4 by a methotrexate-insensitive de novo pathway, and (ii) the conversion of sepiapterin to BH4 by a pterin salvage pathway dependent on dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity. In a Chinese hamster ovary cell mutant lacking dihydrofolate reductase (DUKX-B11), endogenous formation of BH4 proceeds normally but, unlike the parent cells, these cells or extracts of them do not convert sepiapterin or 7,8-dihydrobiopterin to BH4. KB cells, which do not contain detectable levels of GTP cyclohydrolase or BH4 but do contain dihydrofolate reductase, readily convert sepiapterin to BH4 and this conversion is completely prevented by methotrexate. In supernatant fractions of bovine adrenal medulla, the conversion of sepiapterin to BH4 is completely inhibited by methotrexate. Similarly, this conversion in rat brain in vivo is methotrexate-sensitive. Sepiapterin and 7,8-dihydrobiopterin apparently do not enter the de novo pathway of BH4 biosynthesis and may be derived from labile intermediates which have not yet been characterized.

Topics: Adrenal Medulla; Animals; Biopterins; Brain; Cattle; Cells, Cultured; Cricetinae; Guanosine Triphosphate; Methotrexate; Mice; Pteridines; Pterins; Rats

Using Escherichia coli guanosine triphosphate cyclohydrolase, dihydroneopterin triphosphate was synthesized from guanosine triphosphate and was compared with sepiapterin as a substrate for tetrahydrobiopterin formation in bovine adrenal medulla extracts. The dihydrofolate reductase inhibitor, methotrexate, blocks the formation of tetrahydrobiopterin from sepiapterin but not from dihydroneopterin triphosphate. Reduced nicotinamide adenine dinucleotide phosphate and a divalent metal ion are required in partially purified preparations (gel filtration of 40-60% ammonium sulfate fraction on Ultrogel ACA-34) for the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate. Sepiapterin was converted only to dihydrobiopterin in the same fractions since dihydrofolate reductase was removed. The evidence indicates that both dihydroneopterin triphosphate and sepiapterin are good precursors of tetrahydrobiopterin but they are not on the same pathway, contrary to previous proposals.

Topics: Adrenal Medulla; Animals; Biopterins; Cattle; Chemical Phenomena; Chemistry; Guanosine Triphosphate; Methotrexate; Neopterin; Pteridines; Pterins

Since there is no nutritional requirement for the biopterin cofactor, we attempted to create a drug-induced deficiency in rats in order to study the role of tetrahydrobiopterin in regulating the biosynthesis of dopamine and serotonin. The hypothesis that dihydrofolate reductase (EC 1.5.1.3) mediates the final step in the de novo synthesis of tetrahydrobiopterin was tested by treating rats with methotrexate along with leucovorin as a protective agent; there was no reduction in total biopterin or in the fraction present as tetrahydrobiopterin in adrenal medulla, adrenal cortex, pituitary, brain, or pineal glands. Similar results were obtained with metoprine, a lipid-soluble inhibitor of dihydrofolate reductase which readily enters the central nervous system. Treatment with loading doses of phenylalanine along with methotrexate reduced the level of tetrahydrobiopterin in liver. Neuroblastoma N115 cells growing in medium supplemented with thymidine and hypoxanthine continued to form normal amounts of tetrahydrobiopterin in the presence of concentrations of methotrexate which completely inhibited dihydrofolate reductase; higher concentrations of methotrexate increased the tetrahydrobiopterin content of the cells 2-fold and the total biopterin in the medium 3-fold. Although attempts to create a drug-induced deficiency were unsuccessful, the evidence indicates that the de novo synthesis of tetrahydrobiopterin proceeds by a pathway independent of dihydrofolate reductase and that folate antagonists, such as methotrexate are unlikely to impair the hydroxylation of tyrosine and tryptophan, which is dependent upon the availability of the biopterin cofactor.

Topics: Animals; Biopterins; Cell Line; Clone Cells; Folic Acid Antagonists; GTP Cyclohydrolase; Guanosine Triphosphate; Male; Methotrexate; Neuroblastoma; Pteridines; Rats; Rats, Inbred Strains

Topics: Biopterins; Dihydropteridine Reductase; Dopamine; Guanosine Triphosphate; Humans; Phenylpyruvic Acids; Pteridines