Guanosine triphosphate cyclohydrolase activity in rat tissues

Bellahsene, Zina; Dhondt, Jean-Louis; Farriaux, J. P.

doi:10.1042/bj2170059

Cited by 32 publications

(20 citation statements)

References 21 publications

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“…The recombinant rat enzyme could not be distinguished from the enzyme purified from rat liver on the basis of native molecular weight, subunit molecular weight, or ion spray mass spectrometric analysis of the molecular weights of peptides obtained from lysyl-endopeptidase digests of the two enzymes. Consistent with this finding, we determined that GTP cyclohydrolase I purified from rat liver was also not inhibited by BH4, although the enzyme activity in crude liver extracts was inhibited, as reported (7). In order to determine if a dissociable inhibitory factor existed, we fractionated the 100,000g supernatant obtained from rat liver homogenates by gel filtration.…”

supporting

confidence: 78%

“…The activity of GTP cyclohydrolase I in the liver is thought to be regulated by feedback inhibition. The enzyme activity in crude rat liver extracts is inhibited by BH4 (7), and the in vivo occurrence of such feedback inhibition is strongly suggested by clinical observation of patients with an inherited disorder of BH4 metabolism (8). However, the biochemical mechanisms underlying the feedback inhibition of this enzyme by BH4 remain obscure.…”

mentioning

confidence: 99%

“…A mutant TAg protein with a Pro584 --Leu substitution was used because it is expressed in greater amounts than the wild-type TAg (6) and therefore can be detected more readily. We chose an antisense target sequence located 150 nucleotides (nt) downstream of the TAg translation initiation codon because this RNA site had been previously targeted with microinjected antisense RNA (7). The P-gal expression plasmid provided a control for cytostatic and nonspecific effects of the ODNs.…”

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confidence: 99%

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Feedback Regulation Mechanisms for the Control of GTP Cyclohydrolase I Activity

Harada

Kagamiyama

Hatakeyama

1993

Science

150

196

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Guanosine triphosphate (GTP) cyclohydrolase I, the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin (BH4), is subject to feedback inhibition by BH4, a cofactor for phenylalanine hydroxylase. Inhibition was found to depend specifically on BH4 and the presence of another protein (p35). The inhibition occurred through BH4-dependent complex formation between p35 protein and GTP cyclohydrolase I. Furthermore, the inhibition was specifically reversed by phenylalanine, and, in conjunction with p35, phenylalanine reduced the cooperativity of GTP cyclohydrolase I. These findings also provide a molecular basis for high plasma BH4 concentrations observed in patients with hyperphenylalaninemia caused by phenylalanine hydroxylase deficiency.

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confidence: 78%

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confidence: 99%

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confidence: 99%

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Feedback Regulation Mechanisms for the Control of GTP Cyclohydrolase I Activity

Harada

Kagamiyama

Hatakeyama

1993

Science

150

196

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“…Thus, when purified GTP cyclohydrolase I is added to rat liver extracts, the activity is strongly inhibited, giving Ͻ50% recovery of the added activity in assays containing total amounts of protein normally used for GTP cyclohydrolase I activity measurements. 2 The phenomenon of increased GTP cyclohydrolase I activity after heat treatment, which has been described previously (29), suggests that the actual overall recovery of stable GTP cyclohydrolase I activity purified by the procedure described here should be considered to be closer to 25% rather than 65%, still a substantial improvement over a previously described procedure (25). Several factors contribute to this improvement.…”

Section: Table IImentioning

confidence: 93%

Purification and Cloning of the GTP Cyclohydrolase I Feedback Regulatory Protein, GFRP

Milstien

Jaffe

Kowlessur

et al. 1996

Journal of Biological Chemistry

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The activity of GTP cyclohydrolase I, the initial enzyme of the de novo pathway for biosynthesis of tetrahydrobiopterin, the cofactor required for aromatic amino acid hydroxylations and nitric oxide synthesis, is sensitive to end-product feedback inhibition by tetrahydrobiopterin. This inhibition by tetrahydrobiopterin is mediated by the GTP cyclohydrolase I feedback regulatory protein GFRP, previously named p35 (Harada, T., Kagamiyama, H., and Hatakeyama, K. (1993) Science 260, 1507-1510), and L-phenylalanine specifically reverses the tetrahydrobiopterin-dependent inhibition. As a first step in the investigation of the physiological role of this unique mechanism of regulation, a convenient procedure has been developed to co-purify to homogeneity both GTP cyclohydrolase I and GFRP from rat liver. GTP cyclohydrolase I and GFRP exist in a complex which can be bound to a GTP-affinity column from which GTP cyclohydrolase I and GFRP are separately and selectively eluted. GFRP is dissociated from the GTP agarose-bound complex with 0.2 M NaCl, a concentration of salt which also effectively blocks the tetrahydrobiopterin-dependent inhibitory activity of GFRP. GTP cyclohydrolase I is then eluted from the GTP-agarose column with GTP. Both GFRP and GTP cyclohydrolase I were then purified separately to near homogeneity by sequential high performance anion exchange and gel filtration chromatography. GFRP was found to have a native molecular mass of 20 kDa and consist of a homodimer of 9.5-kDa subunits. Based on peptide sequences obtained from purified GFRP, oligonucleotides were synthesized and used to clone a cDNA from a rat liver cDNA library by polymerase chain reaction-based methods. The cDNA contained an open reading frame that encoded a novel protein of 84 amino acids (calculated molecular mass 9665 daltons). This protein when expressed in Escherichia coli as a thioredoxin fusion protein had tetrahydrobiopterin-dependent GTP cyclohydrolase I inhibitory activity. Northern blot analysis indicated the presence of an 0.8-kilobase GFRP mRNA in most rat tissues, the amounts generally correlating with levels of GTP cyclohydrolase I and tetrahydrobiopterin. Thus, mRNA levels were relatively high in liver and kidney and somewhat lower in testis, heart, brain, and lung. These results suggest that GFRP is widely expressed and may play a role in regulating not only phenylalanine metabolism in the liver, but also the production of biogenic amine neurotransmitters as well as nitric oxide synthesis.

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“…Since GTP, but not tetrahydrobiopterin, prevented binding of 1 F1 1, and since binding of NS7 was abolished by tetrahydrobiopterin but not by GTP (see Table l), these ligands must have two different binding sites on the enzyme subunit. That means that tetrahydrobiopterin does not bind competitively to the GTPcyclohydrolase substrate site [8] but to another, regulatory, binding site. The findings that in the presence of UTP, binding of antibody NS7 is totally abolished, and that UTP does not compete with GTP (see Table l), suggest that the two nucleotides do not share the same binding site on the enzyme subunit.…”

Section: Discussionmentioning

confidence: 99%

Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli

Schoedon¹,

Redweik²,

Frank³

et al. 1992

European Journal of Biochemistry

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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 Escherichiu 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 1 has a relatively high apparent K,. The values are slightly different for enzyme purified by GTP-agarose (100 pM) and UTP-agarose (1 10 pM). Low turnover numbers of 12/min and 19/min were calculated for the respective enzyme preparations. GTP-cyclohydrolase-I activity was modulated in V,,, 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 pM tetrahydrobiopterin. UTP was able to reduce the inhibition by tetrahydrobioptcrin. 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.CTP cyclohydrolase I catalyzes the conversion of GTP to dihydroneopterin triphosphate. This is the first step in the multienzyme biosynthesis of the cofactor, tetrahydrobiopterin, of the aromatic amino acid hydroxylases [l]. GTP cyclohydrolase I has been described in many biological systems (for review see [2]

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Guanosine triphosphate cyclohydrolase activity in rat tissues

Cited by 32 publications

References 21 publications

Feedback Regulation Mechanisms for the Control of GTP Cyclohydrolase I Activity

Feedback Regulation Mechanisms for the Control of GTP Cyclohydrolase I Activity

Purification and Cloning of the GTP Cyclohydrolase I Feedback Regulatory Protein, GFRP

Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli

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