Zinc plays a key role in human and bacterial GTP cyclohydrolase I

Auerbach, Günter; Herrmann, Anja; Bracher, A.; Bader, Gerd; Gütlich, Markus; Fischer, Markus; Neukamm, M.; Garrido-Franco, M.; Richardson, Jeffrey Ralph James; Nar, Herbert; Huber, Robert; Bacher, Adelbert

doi:10.1073/pnas.240463497

Cited by 128 publications

(136 citation statements)

References 25 publications

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“…Our data showed that Zn 2+ is essential for the enzyme activity, in agreement with a suggestion from a previous crystallographic study [12]. Zn 2+ was proposed to be involved in the catalytic reaction [12]; this proposition is supported by the results of mutation analysis of the E. coli enzyme [14] and most recently by the observation that mutational replacement of residues predicted to form the Zn 2+ binding centre caused catalytic inactivation and reduced the capacity of the E. coli enzyme to bind zinc [27].…”

Section: Biosynthesis In Vivosupporting

confidence: 92%

“…4, a recent crystallographic study showed that Zn 2+ bound to the active centre of the bacterial and human GCH enzymes, with the conclusion that Zn 2+ participated in the catalytic reaction [12]. Besides Zn 2+ , Ca 2+ at nanomolar concentrations was suggested to activate the enzyme [13].…”

Section: Gch Recognizes Mg-free Gtpmentioning

confidence: 99%

“…Zn 2+ was proposed to be involved in the catalytic reaction [12]; this proposition is supported by the results of mutation analysis of the E. coli enzyme [14] and most recently by the observation that mutational replacement of residues predicted to form the Zn 2+ binding centre caused catalytic inactivation and reduced the capacity of the E. coli enzyme to bind zinc [27]. Although an apo-enzyme for Zn 2+ was not generated by incubating with Zn 2+ chelating agents such as EDTA and TPEN, we succeeded in showing the stimulatory effect of Zn 2+ on the enzyme activity by using a protein refolding assay.…”

Section: Biosynthesis In Vivomentioning

confidence: 99%

“…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 biophysical studies suggest a stimulatory effect of Zn 2+ [12] and Ca 2+ [13] on GCH activity. By crystallographic analysis using purified Escherichia coli enzyme [14], an N-terminally truncated form of the recombinant human enzyme [12], and a stimulatory complex of rat GCH and GFRP induced by phenylalanine [15], Zn 2+ was shown to be bound to the active centre of the homodecameric GCH enzyme.…”

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confidence: 99%

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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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Section: Biosynthesis In Vivosupporting

confidence: 92%

Section: Gch Recognizes Mg-free Gtpmentioning

confidence: 99%

Section: Biosynthesis In Vivomentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…4) (39). In the absence of DAHP, the K m and V max for GTP utilization were 75.52 Ϯ 9.78 M and 46.31 Ϯ 1.23 nmol H 2 NTP/min/mg protein, respectively, in accord with published values (39,43,44). On addition of DAHP, the K m for GTP was modestly increased (100.70 Ϯ 17.34 M), and V max was unchanged (47.48 Ϯ 1.89 nmol H 2 NTP/min/mg protein) as determined by nonlinear regression analysis.…”

Section: Development Of a Novel Kinetic Microplate Assay For Gtpchsupporting

confidence: 88%

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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GTPCyclohydrolaseI

Nar¹

2004

Handbook of Metalloproteins

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GTP cyclohydrolase I (GTPCH) catalyzes the first step in pteridine biosynthesis in Nocardia sp. strain NRRL 5646. This enzyme is important in the biosynthesis of tetrahydrobiopterin (BH 4 ), a reducing cofactor required for nitric oxide synthase (NOS) and other enzyme systems in this organism. GTPCH was purified more than 5,000-fold to apparent homogeneity by a combination of ammonium sulfate fractionation, GTP-agarose, DEAE Sepharose, and Ultragel AcA 34 chromatography. The purified enzyme gave a single band for a protein estimated to be 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was estimated to be 253 kDa by gel filtration, indicating that the active enzyme is a homo-octamer. The enzyme follows Michaelis-Menten kinetics, with a K m for GTP of 6.5 M. Nocardia GTPCH possessed a unique N-terminal amino acid sequence. The pH and temperature optima for the enzyme were 7.8 and 56°C, respectively. The enzyme was heat stable and slightly activated by potassium ion but was inhibited by calcium, copper, zinc, and mercury, but not magnesium. BH 4 inhibited enzyme activity by 25% at a concentration of 100 M. 2,4-Diamino-6-hydroxypyrimidine (DAHP) appeared to competitively inhibit the enzyme, with a K i of 0.23 mM. With Nocardia cultures, DAHP decreased medium levels of NO 2 ؊ plus NO 3 ؊ . Results suggest that in Nocardia cells, NOS synthesis of nitric oxide is indirectly decreased by reducing the biosynthesis of an essential reducing cofactor, BH 4 .

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Zinc plays a key role in human and bacterial GTP cyclohydrolase I

Cited by 128 publications

References 25 publications

GTP cyclohydrolase I utilizes metal‐free GTP as its substrate

GTP cyclohydrolase I utilizes metal‐free GTP as its substrate

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

GTPCyclohydrolaseI

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