6-Pyruvoyl Tetrahydropterin Synthase, An Enzyme With a Novel Type of Active Site Involving Both Zinc Binding and an Intersubunit Catalytic Triad Motif; Site-directed Mutagenesis of the Proposed Active Center, Characterization of the Metal Binding Site and Modelling of substrate Binding

Bürgisser, Daniel; Thöny, Beat; Redweik, U.; Heß, Daniel; Heizmann, Claus W.; Huber, Robert; Nar, Herbert

doi:10.1006/jmbi.1995.0558

Cited by 53 publications

(56 citation statements)

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“…In contrast with NOS, our data demonstrate that Zn 2+ is essential for the activity of the GCH enzyme. In addition to GCH, 6-pyruvoyltetrahydropterin synthase also binds Zn 2+ [33]. Our present study emphasizes the importance of Zn 2+ in nitric oxide production.…”

Section: Biosynthesis In Vivosupporting

confidence: 62%

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: 62%

GTP cyclohydrolase I utilizes metal‐free GTP as its substrate

Suzuki

Kurita

Ichinose

2003

European Journal of Biochemistry

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“…1 B, C. The properties of the PCD mutants clearly confirm the location of the active site in the cleft between the two loops connecting helix a-2 with b-strand 3 and strand 4 with helix a-3. The interaction of other enzymes with the pyrimidine moiety of pterins and folates is mediated by a carboxylate group of Asp or Glu residues as has been shown for dihydrofolate reductase (Bolin et al, 19821, and 6-pyruvoyl-tetrahydropterin synthase (Biirgisser et al, 1995). The active site of PCD is flanked by Asp60, Glu64 and Glu80, but all these residues are not conserved in the homologue PCD from Pseudomonas aeruginosa, which has comparable activity (Zhao et al, 1994).…”

Section: Discussionmentioning

confidence: 95%

Location of the Active Site and Proposed Catalytic Mechanism of Pterin‐4A‐Carbinolamine Dehydratase

Köster

Stier²,

Ficner³

et al. 1996

European Journal of Biochemistry

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Based on the recently solved three-dimensional structure of pterin-4a-carbinolamine dehydratase from rat/human liver the involvement of the proposed active-site residues Glu57, Asp60, His61, His62, Tyr69, His79, Arg87 and Asp88 was examined by site-directed mutagenesis. Most of the mutants showed reduced activity, and only the Glu57-Ala mutant and the His6l+Ala, His62-Ala double mutant were fully devoid of activity. The dissociation constants of quinonoid 6,6-dimethyl-7,8-dihydropterin were significantly increased for binding to the Glu57+Ala, His61-Ala, His62-+Ala single mutants and the His61-Ala, His62-+Ala double mutant, confirming that His61 and His62 are essential for substrate binding and catalysis. The mechanism of dehydration is proposed to involve base catalysis at the N(5)-H group of the substrate by His61.

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“…The catalytic zinc ions (shown as green spheres) are positioned near the equator and toward the outside of the assembly, near the confluence of three protein chains. As with mPTPS, the zinc divalent cation is coordinated by the imidazole side chains of three histidine residues (His (29,35). Interestingly, a second unique dyad, His 25 -Asp 54 , is also present in QueD, which we hypothesize is responsible for promoting the novel retroaldol cleavage to form CPH 4 by additional interactions with Cys 27 .…”

Section: Resultsmentioning

confidence: 93%

“…The active sites of both proteins are composed of a constellation of residues that are contributed by adjacent subunits in the biological assembly (34). The substrate in each active site is bound to a conserved zinc divalent cation via the C1Ј and C2Ј hydroxyl groups and the proteins retain similar binding interactions with the substrate (35). Each active site houses an essential cysteine residue whose activation with a conserved Asp/His dyad is proposed to initiate catalysis (29).…”

mentioning

confidence: 99%

Biochemical and Structural Studies of 6-Carboxy-5,6,7,8-tetrahydropterin Synthase Reveal the Molecular Basis of Catalytic Promiscuity within the Tunnel-fold Superfamily

Miles

Roberts

McCarty

et al. 2014

Journal of Biological Chemistry

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Background:The bacterial homolog, 6-carboxy-5,6,7,8-tetrahydropterin synthase, of the eukaryotic 6-pyruvoyltetrahydropterin synthase enzyme acts on the same substrate but produces different products. Results: Structural and biochemical studies trace the differential reactivity to four residues. Conclusion: Differential reactivity between the enzyme homologs is a result of small changes in the enzyme active site. Significance: This work furthers our understanding of how novel activities may arise from common protein-folds.

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6-Pyruvoyl Tetrahydropterin Synthase, An Enzyme With a Novel Type of Active Site Involving Both Zinc Binding and an Intersubunit Catalytic Triad Motif; Site-directed Mutagenesis of the Proposed Active Center, Characterization of the Metal Binding Site and Modelling of substrate Binding

Cited by 53 publications

References 0 publications

GTP cyclohydrolase I utilizes metal‐free GTP as its substrate

GTP cyclohydrolase I utilizes metal‐free GTP as its substrate

Location of the Active Site and Proposed Catalytic Mechanism of Pterin‐4A‐Carbinolamine Dehydratase

Biochemical and Structural Studies of 6-Carboxy-5,6,7,8-tetrahydropterin Synthase Reveal the Molecular Basis of Catalytic Promiscuity within the Tunnel-fold Superfamily

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