Feedback Regulation Mechanisms for the Control of GTP Cyclohydrolase I Activity

Harada, Toshiaki; Kagamiyama, Hiroyuki; Hatakeyama, Kazuyuki

doi:10.1126/science.8502995

Cited by 150 publications

(211 citation statements)

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“…The effects of GFRP on GT-PCHI occur via phenylalanine-or BH 4 -dependent protein complex formation between GTPCHI and GFRP (12,13). In the absence of GFRP, phenylalanine has no effect on GTPCHI activity, which displays typical allosteric enzyme kinetics with strong positive cooperativity with GTP, the substrate (12,14). Phenylalanine and GFRP reduce the positive cooperativity of GTPCHI and, as a result, stimulate the enzyme's activity in the presence of subsaturating concentrations of GTP.…”

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confidence: 99%

“…The identification of GFRP, which functions as both a positive and negative regulator of GTPCHI, has revealed the tight regulation of GTPCHI activity that maintains intracellular BH 4 levels at and below those needed by BH 4 -requiring enzymes (12). GFRP mediates feed-forward activation of GTPCHI activity by enhancing GTP binding in the presence of phenylalanine while it induces feedback inhibition of enzyme activity in the presence of BH 4 .…”

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confidence: 99%

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Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP

Maita

Okada

Hatakeyama

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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In the presence of phenylalanine, GTP cyclohydrolase I feedback regulatory protein (GFRP) forms a stimulatory 360-kDa complex with GTP cyclohydrolase I (GTPCHI), which is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. The crystal structure of the stimulatory complex reveals that the GTPCHI decamer is sandwiched by two GFRP homopentamers. Each GFRP pentamer forms a symmetrical five-membered ring similar to ␤-propeller. Five phenylalanine molecules are buried inside each interface between GFRP and GTPCHI, thus enhancing the binding of these proteins. The complex structure suggests that phenylalanine-induced GTPCHI⅐GFRP complex formation enhances GTPCHI activity by locking the enzyme in the active state.

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confidence: 99%

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confidence: 99%

Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP

Maita

Okada

Hatakeyama

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…Interestingly, millimolar level concentrations of L-Phe have been shown to reverse this feedback inhibition without causing dissociation of the GFRP⅐GTPCH complex (37). In the absence of BH4, L-Phe triggers GFRP binding to GTPCH resulting in increased GTPCH activity at low levels of GTP substrate (35,38). Because L-Phe is potentially neurotoxic, the ability of L-Phe to stimulate GTPCH activity may have evolved to provide an abundant supply of BH4 cofactor for optimal activity of phenylalanine hydroxylase.…”

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confidence: 99%

“…1) and, inasmuch as GTPCH is regulated by end product feedback inhibition, DAHP could potentially inhibit GTPCH activity by co-opting the endogenous feedback inhibitory mechanism. Feedback inhibition by BH4 is mediated by the formation of a ternary complex between BH4, GTPCH, and a protein termed GTPCH feedback regulatory protein (GFRP) (35,36). GTPCH is a stable decamer, and in the presence of BH4, feedback inhibition is engaged by the binding of one GFRP pentamer to each of the two pentameric faces of GTPCH.…”

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The Mechanism of Potent GTP Cyclohydrolase I Inhibition by 2,4-Diamino-6-hydroxypyrimidine

Kolinsky¹,

Gross

2004

Journal of Biological Chemistry

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Inhibition of GTP cyclohydrolase I (GTPCH) has been used as a selective tool to assess the role of de novo synthesis of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) in a biological system. Toward this end, 2,4-diamino-6-hydroxypyrimidine (DAHP) has been used as the prototypical GTPCH inhibitor. Using a novel real-time kinetic microplate assay for GTPCH activity and purified prokaryote-expressed recombinant proteins, we show that potent inhibition by DAHP is not the result of a direct interaction with GTPCH. Rather, inhibition by DAHP in phosphate buffer occurs via an indirect mechanism that requires the presence of GTPCH feedback regulatory protein (GFRP). Notably, GFRP was previously discovered as the essential factor that reconstitutes inhibition of pure recombinant GTPCH by the pathway end product BH4. Thus, DAHP inhibits GTPCH by engaging the endogenous feedback inhibitory system. We further demonstrate that L-Phe fully reverses the inhibition of GTPCH by DAHP/GFRP, which is also a feature in common with inhibition by BH4/GFRP. These findings suggest that DAHP is not an indiscriminate inhibitor of GTPCH in biological systems; instead, it is predicted to preferentially attenuate GTPCH activity in cells that most abundantly express GFRP and/or contain the lowest levels of L-Phe.(6R)-5,6,7,8-Tetrahydro-L-biopterin (BH4) 1 is an essential cofactor for phenylalanine hydroxylase (1), tyrosine hydroxylase (2), tryptophan hydroxylase (3), glycerol ether monoxygenases (4, 5), and the three isoforms of nitric-oxide synthase (6, 7). Because the activity of these enzymes is typically limited by the availability of BH4, intracellular levels of BH4 determine the rate of production of several key cell-signaling molecules including nitric oxide (NO), dopamine, norepinephrine, epinephrine, and serotonin. In mammalian cells, BH4 levels are primarily dictated by the activity of GTP cyclohydrolase I (GTPCH), the first of three enzymes in the de novo BH4 synthesis pathway (8). Mutations in the GTPCH gene are responsible for severe diseases including 3,4-dehydroxyphenylalanine (dopa)-responsive dystonia (9) and atypical phenylketonuria (10). Other neurological disorders, including Parkinson's disease (11), Alzheimer's disease (12, 13), depression (14), and vascular endothelial dysfunction resulting from diabetes (15, 16), smoking (17-19), and hypercholesterolemia (20), are all associated with deficient BH4 levels. The prototypical GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP) has been used extensively to assess the roles of BH4 and GTPCH in physiological and pathophysiological conditions, although the exact mechanism of this inhibition has not been defined.Four decades ago, DAHP was identified as a potent inhibitor of biopterin-dependent growth of the protozoan Crithidia fasciculata (21). Investigations of biopterin excretion in rats suggested the presence of a de novo biopterin synthesis pathway that is potently inhibited following DAHP treatment (22). Subsequently, biosynthesis of 7,8-dihydrobiopterin from GTP was demons...

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“…At the post-transcriptional level, BH 4 was shown to inhibit, and phenylalanine to stimulate, GCH activity through interaction with GFRP, a GTP cyclohydrolase I feedback regulatory protein [9]. GCH, which is a homodecameric protein, shows positive cooperativity against the GTP substrate [10] and phenylalanine changes the substrate velocity curve from sigmoidal to hyperbolic [11].…”

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GTP cyclohydrolase I utilizes metal‐free GTP as its substrate

Suzuki

Kurita

Ichinose

2003

European Journal of Biochemistry

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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 Mg 2+ , as well as by Mn 2+ , Co 2+ or Zn 2+ , 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 Zn 2+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 Zn 2+ 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 Zn 2+ for its catalytic activity. We suggest that the cellular concentration of divalent cations can modulate GCH activity, and thus tetrahydrobiopterin biosynthesis as well.Keywords: GTP cyclohydrolase I; magnesium; recombinant protein; tetrahydrobiopterin; zinc.Metal ions are essential for many physiological functions of the brain. They may also induce or aggravate numerous neurodegenerative processes. Thus, it is important to understand the roles of metal ions in normal and pathological brain functions.GTP cyclohydrolase I (GCH) is the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH 4 ), and the cellular BH 4 content is regulated mainly by the activity of this enzyme. BH 4 is an essential cofactor for three aromatic amino-acid monooxygenases ) phenylalanine, tyrosine, and tryptophan hydroxylases -and for nitric oxide synthase [1]. BH 4 deficiency caused not only a decrease in the activity of these enzymes but also a decrease in the protein levels of tyrosine hydroxylase and nitric oxide synthase [2,3]. Therefore, the availability of BH 4 affects the amounts of neurotransmitters such as catecholamines, serotonin, melatonin and nitric oxide. The role of BH 4 in the activity of nitric oxide synthase also makes BH 4 an important factor for the immune system and endothelial cell function.Various hormones and cytokines are known to induce the expression of the GCH gene in neural, lymphocytic and endothelial cells, and in different cell lines, resulting in an increased BH 4 content [4][5][6][7][8]. At the post-transcriptional level, BH 4 was shown to inhibit, and phenylalanine to stimulate, GCH activity through interaction with GFRP, a GTP cyclohydrolase I feedback regulatory protein [9]. GCH, which is a homodecameric protein, shows positive cooperativity against the GTP substrate [10] and phenylalanine changes the substrate velocity curve from sigmoidal to hyperbolic [11].Recent...

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Feedback Regulation Mechanisms for the Control of GTP Cyclohydrolase I Activity

Cited by 150 publications

References 39 publications

Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP

Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP

The Mechanism of Potent GTP Cyclohydrolase I Inhibition by 2,4-Diamino-6-hydroxypyrimidine

GTP cyclohydrolase I utilizes metal‐free GTP as its substrate

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