6-methyltetrahydropterin and sapropterin

Tyrosine hydroxylase (TyrH) is a pterin-dependent enzyme that catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine. The oxidation state of the active site iron atom plays a central role in the regulation of the enzyme. The kinetics of reduction of ferric TyrH by several reductants were determined by anaerobic stopped-flow spectroscopy. Anaerobic rapid freeze-quench EPR confirmed that the change in the near-UV absorbance of TyrH upon adding reductant corresponded to iron reduction. Tetrahydrobiopterin reduces wild-type TyrH following a simple second-order mechanism with a rate constant of 2.8 +/- 0.1 mM(-)(1) s(-)(1). 6-Methyltetrahydropterin reduces the ferric enzyme with a second-order rate constant of 6.1 +/- 0.1 mM(-)(1) s(-)(1) and exhibits saturation kinetics. No EPR signal for a radical intermediate was detected. Ascorbate, glutathione, and 1,4-benzoquinone all reduce ferric TyrH, but much more slowly than tetrahydrobiopterin, suggesting that the pterin is a physiological reductant. E332A TyrH, which has an elevated K(m) for tetrahydropterin in the catalytic reaction, is reduced by tetrahydropterins with the same kinetic parameters as those of the wild-type enzyme, suggesting that BH(4) does not bind in the catalytic conformation during the reduction. Oxidation of ferrous TyrH by molecular oxygen can be described as a single-step second-order reaction, with a rate constant of 210 mM(-)(1) s(-)(1). S40E TyrH, which mimics the phosphorylated state of the enzyme, has oxidation and reduction kinetics similar to those of the wild-type enzyme, suggesting that phosphorylation does not directly regulate the interconversion of the ferric and ferrous forms.

Topics: Anaerobiosis; Animals; Ascorbic Acid; Binding Sites; Biopterins; Chromatography, High Pressure Liquid; Electron Spin Resonance Spectroscopy; Iron; Kinetics; Oxidation-Reduction; Pterins; Rats; Spectrophotometry, Ultraviolet; Tyrosine 3-Monooxygenase

In the present study we have analyzed the effect of tetrahydrobiopterin (BH4) essential cofactor for tyrosine hydroxylase and nitric oxide synthase, on the 3,4-dihydroxyphenylalanine (L-DOPA) release from in vitro incubated striatal tissue. dl-6-methyl-5,6,7,8 tetrahydropterine (6-MPH4)-stimulated L-DOPA release in a concentration-dependent manner in the range from 25 to 100 microM. At these concentrations 6-MPH4 did not have any effect on dopamine release. Presence of Nomega-Nitro-L-arginine methyl ester (L-NAME, 200 microM), a nitric oxide synthase inhibitor, but not of alpha-methyl-rho-tyrosine (alpha-MPT, 100 microM), a tyrosine hydroxylase inhibitor, blocked L-DOPA release induced by 6-MPH4 (200 microM). Also, the addition to the incubation medium of melatonin (MEL, 300 microM), which is a scavenger of NO and other free radicals, blocked the L-DOPA release induced by 6-MPH4 (200 microM) but this effect did not occur with the addition of the peroxynitrite scavenger uric acid (UA, 300 microM). Sodium nitroprusside (SNP, 100 muM), a NO generator and l-DOPA releaser as previously reported, potentiated the L-DOPA releasing effect of 6-MPH4 (200 microM) which was also blocked by melatonin. In summary 6-MPH4 stimulates L-DOPA release from striatal fragments incubated in vitro by a mechanism which involves NO or other free radicals derived from NO but not peroxynitrite.

Topics: alpha-Methyltyrosine; Animals; Biopterins; Corpus Striatum; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radical Scavengers; In Vitro Techniques; Levodopa; Male; Melatonin; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Pterins; Rats; Rats, Sprague-Dawley; Tyrosine 3-Monooxygenase

Phenylalanine hydroxylase (PAH) is the key enzyme in the catabolism of L-Phe. The natural cofactor of PAH, 6R-tetrahydrobiopterin (BH4), negatively regulates the enzyme activity in addition to being an essential cosubstrate for catalysis. The analogue 6-methyltetrahydropterin (6M-PH4) is effective in catalysis but does not regulate PAH. Here, the thermodynamics of binding of BH4 and 6M-PH4 to human PAH have been studied by isothermal titration calorimetry. At neutral pH and 25 degrees C, BH4 binds to PAH with higher affinity (Kd = 0.75 +/- 0.18 microM) than 6M-PH4 (Kd = 16.5 +/- 2.7 microM). While BH4 binding is a strongly exothermic process (DeltaH = -11.8 +/- 0.4 kcal/mol) accompanied by an entropic penalty (-TDeltaS = 3.4 +/- 0.4 kcal/mol), 6M-PH4 binding is both enthalpically (DeltaH = -3.3 +/- 0.3 kcal/mol) and entropically (-TDeltaS = -3.2 kcal/mol) driven. No significant changes in binding affinity were observed in the 5-35 degrees C temperature range for both pterins at neutral pH, but the enthalpic contribution increased with temperature rendering a heat capacity change (DeltaCp) of -357 +/- 26 cal/mol/K for BH4 and -63 +/- 12 cal/mol/K for 6M-PH4. Protons do not seem to be taken up or released upon pterin binding. Structure-based energetics calculations applied on the molecular dynamics simulated structures of the complexes suggest that in the case of BH4 binding, the conformational rearrangement of the N-terminal tail of PAH contribute with favorable enthalpic and unfavorable entropic contributions to the intrinsic thermodynamic parameters of binding. The entropic penalty is most probably associated to the reduction of conformational flexibility at the protein level and disappears for the L-Phe activated enzyme. The calculated energetic parameters aid to elucidate the molecular mechanism for cofactor recognition and the regulation of PAH by the dihydroxypropyl side chain of BH4.

Topics: Biopterins; Buffers; Calorimetry; Glucose Oxidase; Humans; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Oxidation-Reduction; Phenylalanine Hydroxylase; Pterins; Thermodynamics; Titrimetry

How 6R-tetrahydrobiopterin (H(4)B) participates in Arg hydroxylation as catalyzed by the nitric oxide synthases (NOSs) is a topic of current interest. Previous work with the oxygenase domain of inducible NOS (iNOSoxy) demonstrated that H(4)B radical formation is kinetically coupled to disappearance of an initial heme-dioxy intermediate and to Arg hydroxylation in a single turnover reaction run at 10 degrees C [Wei, C.-C., Wang, Z.-Q., Wang, Q., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) J. Biol. Chem. 276, 315-319]. Here we used 5-methyl-H(4)B to investigate how pterin structure influences radical formation and associated catalytic steps. In the presence of Arg, the heme-dioxy intermediate in 5-methyl-H(4)B-bound iNOSoxy reacted at a rate of 35 s(-)(1), which is 3-fold faster than with H(4)B. This was coupled to a faster rate of 5-methyl-H(4)B radical formation (40 vs 12.5 s(-)(1)) and to a faster and more productive Arg hydroxylation. The EPR spectrum of the enzyme-bound 5-methyl-H(4)B radical had different hyperfine structure than the bound H(4)B radical and exhibited a 3-fold longer half-life after its formation. A crystal structure of 5-methyl-H(4)B-bound iNOSoxy revealed that there are minimal changes in conformation of the bound pterin or in its interactions with the protein as compared to H(4)B. Together, we conclude the following: (1) The rate of heme-dioxy reduction is linked to pterin radical formation and is sensitive to pterin structure. (2) Faster heme-dioxy reduction increases the efficiency of Arg hydroxylation but still remains rate limiting for the reaction. (3) The 5-methyl group influences heme-dioxy reduction by altering the electronic properties of the pterin rather than changing protein structure or interactions. (4) Faster electron transfer from 5-methyl-H(4)B may be due to increased radical stability afforded by the N-5 methyl group.

Topics: Animals; Arginine; Biopterins; Catalysis; Crystallization; Crystallography, X-Ray; Electron Transport; Free Radicals; Heme; Isoenzymes; Kinetics; Mice; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidation-Reduction; Oxygen; Protein Binding; Pterins; Spectrophotometry, Ultraviolet; Structure-Activity Relationship

Inducible nitric-oxide synthase (NOS) was expressed and purified in the absence of 6(R)-tetrahydro-l-biopterin (H(4)B). Pterin-free NOS exhibits a Soret band (416-420 nm) characteristic of predominantly low spin heme and does not catalyze the formation of nitric oxide (. NO) (Rusche, K. M., Spiering, M. M., and Marletta, M. A. (1998) Biochemistry 37, 15503-15512). Reconstitution of pterin-free NOS with H(4)B was monitored by a shift in the Soret band to 396-400 nm, the recovery of.NO-forming activity, and the measurement of H(4)B bound to the enzyme. As assessed by these properties, H(4)B binding was not rapid and required the presence of a reduced thiol. Spectral changes and recovery of activity were incomplete in the absence of reduced thiol. Full reconstitution of holoenzyme activity and stoichiometric H(4)B binding was achieved in the presence of 5 mm glutathione (GSH). Preincubation with GSH before the addition of H(4)B decreased, whereas lower concentrations of GSH extended, the time required for reconstitution. Six protected cysteine residues in pterin-free NOS were identified by labeling of NOS with cysteine-directed reagents before and after reduction with GSH. Heme and metal content of pterin-free and H(4)B-reconstituted NOS were also measured and were found to be independent of H(4)B content. Additionally, pterin-free NOS was reconstituted with 6-methylpterin analogs, including redox-stable deazapterins. Reconstitution with the redox-stable pterin analogs was neither time- nor thiol-dependent. Apparent binding constants were determined for the 6-methyl- (50 microm) and 6-ethoxymethyl (200 microm) deazapterins. The redox-stable pterin analogs appear to bind to NOS in a different manner than H(4)B.

Topics: Animals; Apoenzymes; Biopterins; Cysteine; Glutathione; Heme; Holoenzymes; Mice; Molecular Structure; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pterins; Spectrophotometry; Zinc

Kinetic studies of tetrameric recombinant human tyrosine hydroxylase isoform 1 (hTH1) have revealed properties so far not reported for this enzyme. Firstly, with the natural cofactor (6R)-Lerythro-5,6,7, 8-tetrahydrobiopterin (H4biopterin) a time-dependent change (burst) in enzyme activity was observed, with a half-time of about 20 s for the kinetic transient. Secondly, nonhyperbolic saturation behaviour was found for H4biopterin with a pronounced negative cooperativity (0.39 < h < 0.58; [S]0.5 = 24 +/- 4 microM). On phosphorylation of Ser40 by protein kinase A, the affinity for H4biopterin increased ([S]0.5 = 11 +/- 2 microM) and the negative cooperativity was amplified (h = 0.27 +/- 0.03). The dimeric C-terminal deletion mutant (Delta473-528) of hTH1 also showed negative cooperativity of H4biopterin binding (h = 0.4). Cooperativity was not observed with the cofactor analogues 6-methyl-5,6,7,8-tetrahydropterin (h = 0.9 +/- 0.1; Km = 62.7 +/- 5.7 microM) and 3-methyl-5,6,7, 8-tetrahydropterin (H43-methyl-pterin)(h = 1.0 +/- 0.1; Km = 687 +/- 50 microM). In the presence of 1 mM H43-methyl-pterin, used as a competitive cofactor analogue to BH4, hyperbolic saturation curves were also found for H4biopterin (h = 1.0), thus confirming the genuine nature of the kinetic negative cooperativity. This cooperativity was confirmed by real-time biospecific interaction analysis by surface plasmon resonance detection. The equilibrium binding of H4biopterin to the immobilized iron-free apoenzyme results in a saturable positive resonance unit (DeltaRU) response with negative cooperativity (h = 0.52-0.56). Infrared spectroscopic studies revealed a reduced thermal stability both of the apo-and the holo-hTH1 on binding of H4biopterin and Lerythro-dihydrobiopterin (H2biopterin). Moreover, the ligand-bound forms of the enzyme also showed a decreased resistance to limited tryptic proteolysis. These findings indicate that the binding of H4biopterin at the active site induces a destabilizing conformational change in the enzyme which could be related to the observed negative cooperativity. Thus, our studies provide new insight into the regulation of TH by the concentration of H4biopterin which may have significant implications for the physiological regulation of catecholamine biosynthesis in neuroendocrine cells.

Topics: Animals; Apoenzymes; Biopterins; Cattle; Enzyme Stability; Humans; Kinetics; Ligands; Mice; Protein Binding; Protein Conformation; Pterins; Rats; Recombinant Proteins; Substrate Specificity; Surface Plasmon Resonance; Temperature; Tyrosine 3-Monooxygenase

Phenylalanine hydroxylase (PAH) purified from rat liver is an oligomeric protein (predominantly tetramers) composed of 52-kDa subunits that are identical in primary structure. We have used radiation target analysis to probe the subunit organization of the enzyme. When 6-methyltetrahydropterin was used as the cofactor, the loss of hydroxylase activity as a function of radiation dose was defined by a single exponential decay, yielding a target size of about 120 kDa. However, when the enzyme was assayed with the natural cofactor tetrahydrobiopterin (BH4), the inactivation curves were much more complex. In these cases, the activity first increased, then decreased, as a function of radiation dose. The inactivation profile at higher radiation doses implied a target size of approximately 100 kDa. Kinetic analysis of the enzyme was significantly activated relative to the nonirradiated sample. In addition, the irradiated enzyme was desensitized to substrate-level activation by phenylalanine. The initial increase in activity at low radiation doses is due to the destruction of a large inhibitor. Analysis of the irradiated samples by high-performance size-exclusion chromatography indicated that the hydroxylase tetramer was lost with a target size of 110 kDa. Our data indicate that the tetrameric form of purified PAH consists of two enzymatically active dimers and that BH4 interacts with a tetramer to inhibit or deactivate the enzymatic activity.

Topics: Animals; Biopterins; In Vitro Techniques; Kinetics; Liver; Molecular Structure; Molecular Weight; Phenylalanine Hydroxylase; Protein Conformation; Pterins; Radiation Dosage; Rats

Recombinant human liver phenylalanine hydroxylase (PAH) expressed in Escherichia coli has been purified to homogeneity. The recombinant enzyme exists in solution as a mixture of 80% tetramers and 20% dimers. A study of the kinetic properties of the enzyme indicates that compared to the recombinant and the native rat liver enzymes, the recombinant human enzyme is in an activated state. This conclusion is supported by the finding that its catalytic activity is only marginally stimulated by incubation with either phenylalanine or lysolecithin. In contrast, the native and the recombinant rat liver enzymes are activated 8- to 25-fold, respectively, when preincubated with phenylalanine or lysolecithin. In the absence of activators, the ratio of the hydroxylase activity in the presence of 6-methyl-5,6,7,8-tetrahydropterin compared to the activity in the presence of (6R)-5,6,7,8-tetrahydrobiopterin (BH4), which is an index of the state of activation of the enzyme, is 4 for the human recombinant PAH compared to a value of 12 for the recombinant rat liver enzyme. Furthermore, the Km for phenylalanine in the presence of BH4 is 0.050 mM, a value that is one-fifth that of the recombinant rat liver enzyme. Covalent modification of the human enzyme by phosphorylation with protein kinase A provides further evidence that the human enzyme is in a substantially activated state. Phosphorylation, which results in the incorporation of 0.6 mol of phosphate/mol of subunit, leads to only a modest activation of 1.5-fold compared to about a 3-fold activation seen after phosphorylation of the native and the recombinant rat liver enzymes. Moreover, the recombinant human liver enzyme is less sensitive than the rat liver enzyme to stimulation by lysolecithin when tryptophan is the substrate. Just as is true for the rat liver enzyme, the apparent Km values for tryptophan and pheylalanine vary with the pterin cofactor employed. The ability of 7-tetrahydrobiopterin (7-BH4) to substitute for the natural cofactor tetrahydrobiopterin has been studied in vitro. The apparent Km for 7-BH4 for the recombinant human enzyme is 0.2 mM and the Km for phenylalanine is 0.05 mM. The hydroxylase reaction is severely inhibited by 7-BH4 in the presence of physiological concentrations of BH4. This inhibition can be overcome by a decrease in the concentration of phenylalanine. The implications of these novel properties of human PAH for phenylalanine homoestasis in man are discussed.

Topics: Animals; Base Sequence; Biopterins; DNA Primers; Enzyme Activation; Enzyme Inhibitors; Escherichia coli; Humans; In Vitro Techniques; Kinetics; Lysophosphatidylcholines; Molecular Sequence Data; Molecular Structure; Phenylalanine; Phenylalanine Hydroxylase; Plasmids; Protein Conformation; Pterins; Rats; Recombinant Proteins

A deficit in the endothelial production of nitric oxide (NO) is associated with the sequelae of reperfusion injury. Because endothelial NO synthesis depends on the cofactor tetra-hydrobiopterin (BH4), we hypothesized that depletion of this cofactor underlies the reduction of endothelium-dependent dilation in reperfusion injury.. After occlusion of the left anterior descending coronary artery of a pig for 60 minutes followed by 90 minutes of reperfusion (ischemia/reperfusion), hearts were removed and the arterioles were isolated, cannulated, pressurized, and placed on an inverted microscope stage. Dose responses to the endothelium-independent dilator sodium nitroprusside and the endothelium-dependent dilators serotonin, A23187, and substance P were obtained under control conditions, after incubation with sepiapterin (intracellularly converted to BH4) or synthetic BH4 6-methyltetrahydropterin (MH4), and again after their washout. After ischemia/reperfusion, sodium nitroprusside maximally dilated arterioles (99 +/- 3%), whereas relaxation to serotonin, A23187, and substance P was significantly reduced (19 +/- 9%, 44 +/- 9%, and 54 +/- 8%, respectively). During incubation with sepiapterin (1 mumol/L) or MH4 (10 mumol/L), endothelium-dependent dilation was significantly enhanced (P < .05), whereas the response to sodium nitroprusside was unaltered. After washout, the vasodilatory responses were not significantly different from the initial ischemia/reperfusion responses. Sepiapterin and MH4 did not affect vasodilatory responses in vessels obtained from nonischemic control hearts. As after ischemia/reperfusion, incubation of control vessels with 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTP cyclohydrolase I, decreased endothelium-dependent vasodilation, which was restored in the presence of sepiapterin or MH4.. These data indicate that exogenous administration of sepiapterin or MH4 restores the response to endothelium-dependent vasodilators in pig coronary arterioles after ischemia/ reperfusion. We therefore conclude that ischemia/reperfusion alters the availability or production of BH4, which contributes to blunted endothelial nitroxidergic vasodilation.

Topics: Animals; Biopterins; Endothelium, Vascular; Enzyme Inhibitors; Hemodynamics; Hypoxanthines; In Vitro Techniques; Myocardial Ischemia; Myocardial Reperfusion Injury; Pteridines; Pterins; Reference Values; Swine; Vasodilation

Nitric oxide synthase (NOS) (EC 1.14.23) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. The complex reaction carried out by NOS, which involves NADPH, O2, and enzyme-bound FAD, FMN, and tetrahydrobiopterin (BH4), has only recently begun to be elucidated. Herein we report the characterization of the pterin requirement of murine macrophage NOS. Although purified NOS activity was not dependent on BH4, activity was significantly enhanced by BH4 in a concentration-dependent fashion. NOS purified in the absence of added BH4 was found to contain substoichiometric concentrations of enzyme-bound pterin, where increased concentrations of bound pterin correlated with an increase in activity when assayed in the absence of exogenous BH4. However, NOS purified in the presence of BH4 followed by gel filtration exhibited a 1 mol of pterin:1 mol of NOS 130-kDa subunit stoichiometry and activity that was essentially independent of exogenous BH4. Experiments to probe a redox role for the pterin were carried out using pterin analogues. 6(R,S)-Methyltetrahydropterin was found to increase NOS activity in enzyme purified in the absence of BH4. However, the deaza analogue, 6(R,S)-methyl-5-deazatetrahydropterin, was not only incapable of supporting enzymatic turnover but also inhibited citrulline formation in a concentration-dependent manner. Overall, these results support a role for BH4 in the NOS reaction that involves stabilization of the enzyme and redox chemistry wherein a 1:1 stoichiometry between bound pterin and NOS subunit results in maximum activity.

Topics: Amino Acid Oxidoreductases; Animals; Biopterins; Cells, Cultured; Chromatography, High Pressure Liquid; Macrophages; Mice; Nitric Oxide Synthase; Oxidation-Reduction; Pterins; Spectrometry, Fluorescence

In the presence of tyrosine, phenylalanine hydroxylase, which has been activated with lysolecithin, catalyzes the oxidation of tetrahydrobiopterin at a rate 10-20% that of the parallel reaction with phenylalanine. Unlike the reaction with phenylalanine, there is no net concomitant hydroxylation of tyrosine, although the amino acid is still a necessary component. Tyrosine appears to form an abortive complex with the activated enzyme, the pterin cofactor and molecular oxygen. The Km for tetrahydrobiopterin is identical for the reactions with phenylalanine and tyrosine, whereas the Km for tyrosine is approximately 3 1/2 times greater than the Km for phenylalanine. The tyrosine-dependent oxidation of tetrahydrobiopterin proceeds at both pH 6.8 and 8.2 and shows a similar dependence on the pH as that of the physiological reaction. Tetrahydrobiopterin can be replaced by the artificial cofactor, 6-methyltetrahydropterin, in the tyrosine-dependent oxidation at both pH 6.8 and 8.2. As in the parallel reaction with phenylalanine, both the Km for the cofactor and the Km for the aromatic amino acid increase with this substitution.

Topics: Animals; Biopterins; Enzyme Activation; Kinetics; Liver; Lysophosphatidylcholines; Oxidation-Reduction; Phenylalanine Hydroxylase; Pterins; Rats; Tyrosine

Pterins inhibit rat liver GTP cyclohydrolase I activity noncompetitively. Reduced pterins, such as 7,8-dihydro-D-neopterin, (6R,S)-5,6,7,8-tetrahydro-D-neopterin, 7,8-dihydro-L-biopterin, (6R)-5,6,7,8-tetrahydro-L-biopterin, L-sepiapterin, and DL-6-methyl-5,6,7,8-tetrahydropterin are approximately 12-times more potent as inhibitors than are oxidized pterins, such as D-neopterin, L-biopterin, and isoxanthopterin. They are also 12-times more potent than folates, such as folic acid, dihydrofolic acid, (+/-)-L-tetrahydrofolic acid, and aminopterin. The Ki values for 7,8-dihydro-D-neopterin, 7,8-dihydro-L-biopterin, and (6R)-5,6,7,8-tetrahydro-L-biopterin are 12.7 microM, 14.4 microM, and 15.7 microM, respectively. These results suggest that mammalian GTP cyclohydrolase I may be regulated by its metabolic end products.

Topics: Aminohydrolases; Animals; Biopterins; Folic Acid; GTP Cyclohydrolase; Kinetics; Liver; Neopterin; Oxidation-Reduction; Pteridines; Pterins; Rats; Xanthopterin

The effects of (6R)- and (6S)-tetrahydrobiopterin (BPH4), tetrahydroneopterin, and 6-methyltetrahydropterin on the activity of tryptophan hydroxylase were investigated in rat raphe slices. The activity of tryptophan hydroxylase was estimated by measurement of 5-hydroxytryptophan (5-HTP) formation under inhibition of aromatic L-amino acid decarboxylase with use of HPLC-fluorometric detection. (6R)-BPH4 (the naturally occurring form) at 42 microM, tetrahydroneopterin at 50 microM, and 6-methyltetrahydropterin at 100 microM increased tryptophan hydroxylase activity to 350, 145, and 146% of control values, respectively. (6S)-BPH4, however, had no significant effects on tryptophan hydroxylase activity. These results suggest that tryptophan hydroxylase is subsaturating in vivo for the naturally occurring cofactor, (6R)-BPH4, and that the concentration of (6R)-BPH4 may play an important role for the regulation of tryptophan hydroxylase activity in vivo.

Topics: 5-Hydroxytryptophan; Animals; Biopterins; Male; Pterins; Raphe Nuclei; Rats; Rats, Inbred Strains; Tryptophan Hydroxylase

5,6,7,8-Tetrahydrobiopterin, the naturally occurring essential cofactor for the enzymatic hydroxylations of phenylalanine, tyrosine and tryptophan, and its synthetic analog 2-amino-6-methyl-5,6,7,8-tetrahydro-4(3H)-pteridinone, have been synthesized in good yield by the direct hydrogenation of 1-(2-amino-1,6-dihydro-5-nitro-6-oxopyrimidin-4-yl-amino)-1,5-dide oxy-L- erythro-pentulose and 2-amino-6-hydroxy-5-phenylazo-4-pyrimidylamino-acetone, respectively. The reactions were carried out at room temperature in trifluoroacetic acid over a platinum catalyst at 2 atm and the products, each containing a mixture of the two possible C-6 isomers, were isolated by precipitation. The simplicity of the preparative method suggests the procedure may be applied generally to the synthesis of all tetrahydropteridines derived from similar pyrimidine precursors.

Topics: Biopterins; Chemical Phenomena; Chemistry; Isomerism; Pteridines; Pterins; Temperature

A patient with hyperphenylalaninemia caused by a defect in the synthesis of tetrahydrobiopterin was treated with 6-methyltetrahydropterin. This synthetic analog of the naturally occurring hydroxylation cofactor tetrahydrobiopterin, when given orally at a daily dose of 20 mg per kilogram of body weight increased depressed plasma and cerebrospinal fluid levels of norepinephrine. At a daily dose of 8 mg/kg, this pterin increased depressed cerebrospinal fluid levels of the biogenic amine metabolites dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid. At these doses of 6-methyltetrahydropterin, there was an improvement of the patient's neurological symptoms, including a pronounced decrease in eye rolling and drooling and a marked increase in muscle strength, coordination, and physical activity.

Topics: Biopterins; Blood Glucose; Child; Dose-Response Relationship, Drug; Growth Hormone; Humans; Male; Neurotransmitter Agents; Phenylalanine; Phenylketonurias; Prolactin; Pteridines; Pterins

The literature establishes a potential link between the function of tetrahydroisoquinolines in the biological actions of alcohol and the reduced pterin cofactor needed for synthesis of certain neurotransmitters. The effect of the administration of 6-methyltetrahydropterin and tetrahydrobiopterin upon alcohol withdrawal in mice was tested. Dependence was produced by ip administration of alcohol, supplemented by alcohol delivered by a subcutaneously implanted silastic tube. Administration of the reduced pterins at relatively high doses did not reduce the magnitude of the alcohol withdrawal syndrome.

Topics: Alcoholism; Animals; Biopterins; Ethanol; Female; Humans; Mice; Pteridines; Pterins; Substance Withdrawal Syndrome

The properties of purified tyrosine hydroxylase (TH) from bovine corpus striatum, both native and phosphorylated forms of the enzyme, were studied. TH had a tendency toward greater affinity for tetrahydrobiopterin (BH4) than for the synthetic cofactor 6-methyltetrahydropterin (6-MPH4), although the maximal velocity of the TH-catalyzed reaction was greater with 6-MPH4. Phosphorylation increased the affinity of TH for cofactor at pH 6.0, with little change in Vmax. At pH 7.0, phosphorylation caused increased activation of TH by increasing Vmax as well as reducing the Km for cofactor. The K1 for dopamine was increased twofold by phosphorylation at pH 6.0, but eightfold at pH 7.0. Phosphorylation was not associated with a change in Km for tyrosine at any pH or with any cofactor studied, although the Km for tyrosine of TH was cofactor-dependent and seven to eight times greater with 6-MPH4 than with BH4 as cofactor. Heparin and NaCl activated native TH at pH 6.0, but not at pH 7.0. Phosphorylated TH was unaffected by heparin or salt at pH 6.0, but was relatively inhibited at pH 7.0. The data are presented in the context of the physiological environment of TH.

Topics: Animals; Biopterins; Cattle; Corpus Striatum; Hydrogen-Ion Concentration; Kinetics; Phosphorylation; Pterins; Structure-Activity Relationship; Substrate Specificity; Tyrosine; Tyrosine 3-Monooxygenase

Substantial amounts of tetrahydrobiopterin and 6-methyltetrahydropterin can be detected in CSF when these pterins are given peripherally to patients with hyperphenylalaninemia due to defective biopterin synthesis. Results of this study suggest that administration of either of these pterins in proper doses may prove to be a treatment not only for the impaired peripheral phenylalanine metabolism, but also for the neurologic disorders that are characteristic of the variant forms of hyperphenylalaninemia due to defective tetrahydrobiopterin synthesis or metabolism.

Topics: Biopterins; Brain; Child; Child, Preschool; Female; Humans; Male; Neopterin; Phenylalanine; Phenylketonurias; Pteridines; Pterins

The content of tetrahydrobiopterin in rat brain was doubled by peripherally administered tetrahydrobiopterin, with the natural 1 diastereoisomer more effective than the unnatural d configuration. The model pteridine, 6-methyltetrahydropterin was ten times more efficient than tetrahydrobiopterin in crossing the blood-brain barrier, and striatal concentrations of 6-methyltetrahydropterin remained elevated for 2 hours, declining with a half-life of 3 hours. While no evidence for a specific uptake mechanism for concentrating 6-methyltetrahydropterin in cells containing tetrahydrobiopterin was detected, the pterin was found in ts presumed site of action, the nerve terminal. Replacement therapy with reduced pterins may therefore be effective in the treatment of the neurological disorders associated with the variant forms of hyperphenylalaninemia that result from defects in the biosynthesis or metabolism of tetrahydrobiopterin within the central nervous system.

Topics: Animals; Biopterins; Blood-Brain Barrier; Brain; Male; Pteridines; Pterins; Rats; Stereoisomerism; Structure-Activity Relationship