Tetrahydrobiopterin and Biogenic Amine Metabolism in the hph-1 Mouse

Abstract: hph-1 mice, which have defective tetrahydrobiopterin biosynthesis due to decreased GTP cyclohydrolase I activity, have been used to investigate the effects of tetrahydrobiopterin deficiency on aromatic l -amino acid monooxygenases and brain monoamine metabolism. Liver tetrahydrobiopterin levels were decreased, and tetrahydrobiopterin deficiency and reduced levels of dopamine, norepinephrine, serotonin, and their metabolites in the brain occurred both pre- and postnatally. Chronic subcutaneous tetrahydrobiopterin elevated brain levels to values higher than those seen in controls but had no effect on monoamine metabolism. In vivo activities of tyrosine hydroxylase and tryptophan hydroxylase were significantly decreased. There was a 30% decrease in the in vitro activity of striatal tyrosine hydroxylase and 50% decrease in liver phenylalanine hydroxylase. Western blotting demonstrated that the lower monooxygenase activities resulted from a reduced absolute amount of tyrosine hydroxylase and phenylalanine hydroxylase protein. The findings suggest involvement of tetrahydrobiopterin in the control of the steady-state concentration of the aromatic l -amino acid monooxygenases. In addition, demonstration of central monoamine changes in the hph-1 mouse make it a possible model system for the investigation of the neuropathological mechanisms in Dopa-responsive dystonia, which has recently been linked with mutations in the gene for GTP cyclohydrolase I.     

Lack of dependence of amine or prostaglandin biosynthesis on absolute cerebral level of pteridine cofactor

Using 2-amino-6-(5'-2'-deoxyphosphoribosyl)-amino-5- or -6-formamido-6-hydroxypyrimidine (dFPyd-P₃), a specific inhibitor of tetrahydrobiopterin (BH₄) synthesis, cerebral pools of BH₄ were reduced to half of that of controls; while, simultaneously, the biosynthesis $\text{de novo}$ of L-erythrodihydroniopterin (BH₂) from GTP was inhibited by about 98%. Nevertheless, there was no effect on the cerebral levels of serotonin, dopamine, norepinephrine or on the biosynthesis of prostaglandin E₂ or F₁. The data are presented in evidence that the absolute level of the cofactor (BH₄) is not regulatory of amine or protaglandin biosynthesis $\text{in vivo}$. Amine and prostaglandin biosynthesis proceeded even at cofactor concentrations of 9×10⁷ M nsuggesting that their biosynthesis is dependent on the rate of H⁺ + e shuttle between BH₂ and BH₄.     

Molecular Biology of Catecholamine-Related Enzymes in Relation to Parkinson's Disease

1. Catecholamine (dopamine, norepinephrine, and epinephrine) biosynthesis is regulated by tyrosine hydroxylase (TH). TH activity is regulated by the concentration of the cofactor tetrahydrobiopterin (BH4), whose level is regulated by GTP cyclohydrolase I (GCH) activity. Thus, GCH activity indirectly regulates TH activity and catecholamine levels.2. TH activity in the nigrostriatal dopaminergic neurons is most sensitive to the decrease in BH4.3. Mutations of GCH result in reductions in GCH activity, BH4, TH activity, and dopamine, causing either recessively inherited GCH deficiency or dominantly inherited hereditary progressive dystonia [HPD; Segawa's disease; also called dopa-responsive dystonia (DRD)].4. In juvenile parkinsonism and Parkinson's disease, which have dopamine deficiency in the basal ganglia as HPD/DRD, the GCH gene may be normal, and the molecular mechanism of the dopamine deficiency in the basal ganglia is different from that in HPD/DRD.     

Regulation of tryptophan and tyrosine hydroxylase

The synthesis of the neurotransmitters serotonin, norepinephrine, and the dopamine is regulated by the initial amino acid hydroxylases. Little is known about the factors that regulate the level of tryptophan hydroxylase in tissue. However, the level of tyrosine hydroxylase is regulated by transsynaptic induction. Acute regulation of in vivo hydroxylase activity appears to be by substrate availability in the case of tryptophan hydroxylase and possibly by feedback inhibition with tyrosine hydroxylase. A newly described phenomenon which has been termed “receptor mediated feedback inhibition” involving neuronal feedback regulation of the activity of both tyrosine and tryptophan hydroxylase may also have an important role.     

Evidence for conformational changes in elongation factor Tu induced by GTP and GDP

A comparative study of the rates of tritium-hydrogen exchange in three liganded states of the protein elongation factor Tu (EFTu) reveals a substantial conformational difference between the free (EFTu) or GTP-bound (EFTu·GTP) forms and when GDP is present (EFTu·GDP). This conformational difference is acentuated with short time tritiations. There are 25–35% more very slow hydrogens in EFTu·GDP than in EFTu·GTP, indicating that $\text{GDP}$ induces a $\text{tighter}$ conformation in EFTu than does $\text{GTP}$. Thus, a rationale is provided for the difference in reactivity of EFTu·GTP and EFTu·GDP for AA-tRNA and a conformational role in regulating protein biosynthesis may be proposed for GTP and GDP. Finally, we demonstrate that nucleoside polyphosphates may cause size-able conformational changes in proteins.     

Regulation of Guanosine Triphosphate Cyclohydrolase and Tetrahydrobiopterin Levels and the Role of the Cofactor in Tyrosine Hydroxylation in Primary Cultures of Adrenomedullary Chromaffin Cells

Selective modification of the tetrahydrobiopterin levels in cultured chromaffin cells were followed by changes in the rate of tyrosine hydroxylation. Addition of sepiapterin, an intermediate on the salvage pathway for tetrahydrobiopterin synthesis, rapidly increased intracellular levels of tetrahydrobiopterin and elevated the rate of tyrosine hydroxylation in the intact cell. Tyrosine hydroxylation was also enhanced when tetrahydrobiopterin was directly added to the incubation medium of intact cells. When the cultured chromaffin cells were treated for 72 h with N-acetylserotonin, an inhibitor of sepiapterin reductase, tetrahydrobiopterin content and the rate of tyrosine hydroxylation were decreased. Addition of sepiapterin or N-acetylserotonin had no consistent effect on total extractable tyrosine hydroxylase activity or on catecholamine content in the cultured chromaffin cells. Three-day treatment of chromaffin cell cultures with compounds that increase levels of cyclic AMP (forskolin, cholera toxin, theophylline, dibutyryl- and 8-bromo cyclic AMP) increased total extractable tyrosine hydroxylase activity and GTP-cyclohydrolase, the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Tetrahydrobiopterin levels and intact cell tyrosine hydroxylation were markedly increased after 8-bromo cyclic AMP. The increase in GTP-cyclohydrolase and tetrahydrobiopterin induced by 8-bromo cyclic AMP was blocked by the protein synthesis inhibitor cycloheximide. Agents that deplete cellular catecholamines (reserpine, tetrabenazine, and brocresine) increased both total tyrosine hydroxylase and GTP-cyclohydrolase activities, although treating the cultures with reserpine or tetrabenazine resulted in no change in cellular levels of cyclic AMP. Brocresine and tetrabenazine increased tetrahydrobiopterin levels, but the addition of reserpine to the cultures decreased catecholamine and tetrahydrobiopterin content and resulted in a decreased rate of intact cell tyrosine hydroxylation in spite of the increased activity of the total extractable enzyme. These data indicate that in cultured chromaffin cells GTP-cyclohydrolase activity like tyrosine hydroxylase activity is regulated by both cyclic AMP-dependent and cyclic AMP-independent mechanisms and that the intracellular level of tetrahydrobiopterin is one of the many factors that control the rate of tyrosine hydroxylation.     

GTP cyclohydrolase I gene,tetrahydrobiopterin, and tysorine hydroxylase gene: Their relations to dystonia and parkinsonism

Catecholamine biosynthesis is regulated by tyrosine hydroxylase (TH) requiring tetrahydrobiopterin (BH4) as the cofactor. We found four (human TH type 1–4) and two isoforms (TH type 1 and 2) in humans and monkeys, while non-primate animals have a single TH corresponding to human TH type 1. BH4 is synthesized from GTP, and GTP cyclohydrolase I (GCH) is the first and regulatory enzyme. Mutations in GCH gene were found to cause both GCH deficiency with autosomal recessive trait and hereditary progressive dystonia with marked diurnal fluctuation (HPD) (Segawa's disease)/or DOPA-responsive dystonia (DRD) with autosomal dominant trait. When GCH activity is decreased to less than 20% of the normal value, the activity of TH in the nigrostriatal dopaminergic neurons may be first decreased resulting in decreases in TH activity and dopamine level, and in the symptoms of HPD/DRD. In contrast to HPD/DRD, juvenile parkinsonism (JP) have normal GCH activity. In Parkinson's disease (PD), GCH, TH, and dopamine in the striatum may decrease in parallel, as the secondary effects caused by cell death. Special issue dedicated to Dr. Kinya Kuriyama.     

Regulation of pteridine-requiring enzymes by the cofactor tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is synthesized from guanosine triphosphate (GTP) by GTP cyclohydrolase I (GCH), 6-pyruvoyltetrahydropterin synthase (PTS), and sepiapterin reductase (SPD). GCH is the rate-limiting enzyme. BH4 is a cofactor for three pteridine-requiring monooxygenases that hydroxylate aromatic L-amino acids, i.e., tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH), and phenylalanine hydroxylase (PAH), as well as for nitric oxide synthase (NOS). The intracellular concentrations of BH4, which are mainly determined by GCH activity, may regulate the activity of TH (an enzyme-synthesizing catecholamines from tyrosine), TPH (an enzyme-synthesizing serotonin and melatonin from tryptophan), PAH (an enzyme required for complete degradation of phenylalanine to tyrosine, finally to CO2 + H2O), and also the activity of NOS (an enzyme forming NO from arginine), Dominantly inherited hereditary progressive dystonia (HPD), also termed DOPA-responsive dystonia (DRD) or Segawa's disease, is a dopamine deficiency in the nigrostriatal dopamine neurons, and is caused by mutations of one allele of the GCH gene. GCH activity and BH4 concentrations in HPD/DRD are estimated to be 2-20% of the normal value. By contrast, recessively inherited GCH deficiency is caused by mutations of both alleles of the GCH gene, and the GCH activity and BH4 concentrations are undetectable. The phenotypes of recessive GCH deficiency are severe and complex, such as hyperphenylalaninemia, muscle hypotonia, epilepsy, and fever episode, and may be caused by deficiencies of various neurotransmitters, including dopamine, norepinephrine, serotonin, and NO. The biosynthesis of dopamine, norepinephrine, epinephrine, serotonin, melatonin, and probably NO by individual pteridine-requiring enzymes may be differentially regulated by the intracellular concentration of BH4, which is mainly determined by GCH activity. Dopamine biosynthesis in different groups of dopamine neurons may be differentially regulated by TH activity, depending on intracellular BH4 concentrations and GCH activity. The nigrostriatal dopamine neurons may be most susceptible to a partial decrease in BH4, causing dopamine deficiency in the striatum and the HPD/DRD phenotype.     

In vitro translation of mRNA from rat pheochromocytoma tumors, characterization of tyrosine hydroxylase

Studies are presented which demonstrate that rat pheochromocytoma tumors are a convenient material for the preparation of tyrosine hydroxylase mRNA. Total pheochromocytoma poly(A)⁺mRNA has been extracted from tumors, then translated in a reticulocyte lysate cell-free system. Neo-synthesized tyrosine hydroxylase has been identified by direct immunoprecipitation followed by sodium dodecyl sulfate acrylamide gel electrophoresis. The proportion of this specific mRNA has been calculated; it represents 0.15 per cent of the total poly(A)⁺mRNA. The molecular weight of the $\text{in}\text{vitro}$ synthesized tyrosine hydroxylase is 62,000.     

Induction of tyrosine hydroxlase synthesis in rat superior cervical ganglia in vitro by nerve growth factor and dexamethasone.

The addition of dexamethasone and nerve growth factor to organ cultures of superior cervical ganglia from young rats induces the synthesis of tyrosine hydroxylase. The combination of nerve growth factor and dexamethasone $\text{in vitro}$ produces a differential rate of tyrosine hydroxylase synthesis which approaches that obtained by the $\text{in vivo}$ administration of nerve growth factor.     

Dopamine inhibition of tryptophan hydroxylase in molluscan nervous tissue homogenates: Evidence for intracellular site of action

PCPA, dopamine and the dopamine agonist epinine inhibited trytophan hydroxylase activity in nervous tissue homogenates of $\text{Anodonta cygnea}$ and $\text{Mytilus edulis}$ (Bivalvia). Haloperidol did not affect tryptophan hydroxylase activity in the homogenates nor did it antagonize dopamine action.     

Inactivation of Tryptophan Hydroxylase by Nitric Oxide: Enhancement by Tetrahydrobiopterin

Abstract: Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is inactivated by the nitric oxide generators sodium nitroprusside, diethylamine/nitric oxide complex, and S -nitroso- N -acetylpenicillamine. Physiological concentrations of tetrahydrobiopterin, the natural and endogenous cofactor for the hydroxylase, significantly enhance the inactivation of the enzyme caused by each of these nitric oxide generators. The substrate tryptophan does not have this effect. The chemically reduced (tetrahydro-) form of the pterin is required for the enhancement, because neither biopterin nor dihydrobiopterin is effective. The 6 S -isomer of tetrahydrobiopterin, which has little cofactor efficacy for tryptophan hydroxylase, does not enhance enzyme inactivation as does the natural 6 R -isomer. A number of synthetic, reduced pterins share with tetrahydrobiopterin the ability to enhance nitric oxide-induced inactivation of tryptophan hydroxylase. The tetrahydrobiopterin effect is not prevented by agents known to scavenge hydrogen peroxide, superoxide radicals, peroxynitrite anions, hydroxyl radicals, or singlet oxygen. On the other hand, cysteine partially protects the enzyme from both the nitric oxide-induced inactivation and the combined pterin/nitric oxide-induced inactivation. These results suggest that the tetrahydrobiopterin cofactor enhances the nitric oxide-induced inactivation of tryptophan hydroxylase via a mechanism that involves attack on free protein sulfhydryls. Potential in vivo correlates of a tetrahydrobiopterin participation in the inactivation of tryptophan hydroxylase can be drawn to the neurotoxic amphetamines.     

Relaxation kinetic studies of coenzyme binding to glutamate dehydrogenase from beef liver.

The enzyme, D-erythrodihydroneopterin triphosphate synthetase from rat brain was observed to have a significantly lower specific activity than that from liver due to their degree of dephosphorylation during preparation. The brain enzyme could be phosphorylated $\text{in vitro}$ in presence of [³²P]-ATP and protein kinase, resulting in an increased specific activity. Isolation of brain enzyme in presence of 0.8 M NaF allowed recovery of the enzyme phosphorylated at residue 67 (serine) as determined by a new assay for phosphate. This enzyme is present in synaptosomes and its state of phosphorylation may regulate the rate at which dihydrobiopterin, the precursor of the hydroxylase cofactor (tetrahydrobiopterin, BH₄), is synthesized by synaptosomes.     

Calcium activation of brain tryptophan hydroxylase.

Using both radioisotopic and fluorometric techniques to measure the activity of midbrain soluble enzyme, we have demonstrated that calcium activates tryptophan hydroxylase. The observed activation apparently results from an increased affinity of the enzyme for both its substrate, tryptophan, and the cofactor 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine (6-MPH₄). The calcium activation of tryptophan hydroxylase appears to be specific for both enzyme and effector: other brain neurotransmitter biosynthetic enzymes, such as aromatic amino acid decarboxylase(s) and tyrosine hydroxylase, are not affected by calcium (at concentrations ranging from 0.01 mM to 2.0 mM); other divalent cations, such as Ba⁺⁺, Mg⁺⁺, and Mn⁺⁺, have no activating effect on tryptophan hydroxylase. This work suggests that increases in brain serotonin biosynthesis induced by neural activation may be due to influx of Ca⁺⁺ associated with membrane depolarization and resulting activation of nerve ending tryptophan hydroxylase.     

The effect of l-ascorbate on catecholamine biosynthesis (Short Communication)

l-Ascorbate stimulates the enzymic hydroxylation of phenylalanine in vitro by recycling tetrahydrobiopterin, which reduces O₂ utilized in the reaction. It is suggested that ascorbate might have a similar function in vivo; this would explain the apparent regulation of tyrosine hydroxylase and tryptophan hydroxylase activities by this vitamin.     

Regulatory effects of purine ribonucleotides on Escherichia coli adenylosuccinate synthase

Adenylosuccinate synthase (EC 6.3.4.4.) ($\text{l-aspartate + GTP + IMP}\text{→}\text{Mg}{}^{\mathrm{2+}}\text{adenylosuccinate + GDP + P}{}_{\mathrm{i}}$) is an important site for the regulation of adenylate biosynthesis. A partially purified preparation of the enzyme from Escherichia coli B showed feedback inhibition by ADP and AMP, weak positive response to the adenylate energy charge, and weak positive response to the mole fraction of GTP in the GTP + GDP pool. These responses seem to ensure that the synthesis of adenine nucleotides will be controlled appropriately in response to the level of end products and to the energy state of the cell, and to avoid the potential difficulties arising from the fact that the end products of this sequence and the indicators of the energy state of the cell are the same compounds.     

Caffeine injection raises brain tryptophan level,but does not stimulate the rate of serotonin synthesis in rat brain

Acute caffeine injection (100 mg/kg) elevates brain levels of tryptophan (TRP), serotonin (5HT), and 5-hydroxyindoleacetic acid (5HIAA). Experiments were performed to determine if the increases in 5HT and 5HIAA result from a stimulation of the rate of 5HT synthesis. Both the rate of 5-hydroxytryptophan (5HTP) accumulation following NSD-1015 injection, and the rate of ³H-5-hydroxyindole synthesis from ³H-tryptophan were measured $\text{in vivo}$ following caffeine administration and found to be normal. Tryptophan hydroxylase activity, as measured $\text{in vitro}$ in brain homogenates, was also unaffected by caffeine. The results suggest that the elevations in brain 5HT and 5HIAA levels produced by caffeine do $\text{not}$ reflect enhanced 5HT synthesis, despite significant elevations in brain TRP level. Some other mechanism(s) must therefore be responsible for these elevations in brain 5-hydroxyindole levels.     

Direct binding of GTP cyclohydrolase and tyrosine hydroxylase: regulatory interactions between key enzymes in dopamine biosynthesis

The signaling functions of dopamine require a finely tuned regulatory network for rapid induction and suppression of output. A key target of regulation is the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, which is activated by phosphorylation and modulated by the availability of its cofactor, tetrahydrobiopterin. The first enzyme in the cofactor synthesis pathway, GTP cyclohydrolase I, is activated by phosphorylation and inhibited by tetrahydrobiopterin. We previously reported that deficits in GTP cyclohydrolase activity in Drosophila heterozygous for mutant alleles of the gene encoding this enzyme led to tightly corresponding diminution of in vivo tyrosine hydroxylase activity that could not be rescued by exogenous cofactor. We also found that the two enzymes could be coimmunoprecipitated from tissue extracts and proposed functional interactions between the enzymes that extended beyond provision of cofactor by one pathway for another. Here, we confirm the physical association of these enzymes, identifying interacting regions in both, and we demonstrate that their association can be regulated by phosphorylation. The functional consequences of the interaction include an increase in GTP cyclohydrolase activity, with concomitant protection from end-product feedback inhibition. In vivo, this effect would in turn provide sufficient cofactor when demand for catecholamine synthesis is greatest. The activity of tyrosine hydroxylase is also increased by this interaction, in excess of the stimulation resulting from phosphorylation alone. Vmax is elevated, with no change in Km. These results demonstrate that these enzymes engage in mutual positive regulation.     

Pyrimidines as cofactors for phenylalanine hydroxylase

It has been generally assumed that a tetrahydropterin (2-amino-5,6,7,8-tetrahydro-4-pteridinone) is essential for activity of the three aromatic amino acid hydroxylases. In this report it is shown that appropriately substituted pyrimidines can assume the role of cofactor for phenylalanine hydroxylase. 2,5,6-Triamino-4-pyrimidinone(V) and 5-benzylamino-2,6-diamino-4-pyrimidinone(VI) possess the same K_m values (0.1 mM and 0.003 mM) and stoichiometry of tyrosine generated to cofactor consumed (0.4 and 1.0) as their corresponding pteridine analogs, tetrahydropterin(III) and 6-phenyltetrahydropterin(IV). However, the rates with pyrimidines are lower. The ratio of rates $\text{V}\text{III}\text{= 0.045}$ and $\text{VI}\text{IV}\text{= 0.015}$. These results indicate that pteridine carbons 6 and 7 are not fundamental to cofactor binding or function, though they markedly influence the maximum velocity of hydroxylation. Pyrimidine cofactors of phenylalanine hydroxylase are valuable probes for the elucidation of the binding forces, transition states, and mechanism of oxygen activation of these hydroxylases.