Tetrahydrobiopterin Inhibits Monomerization and Is Consumed during Catalysis in Neuronal NO Synthase

Reif, Andreas; Fröhlich, L.; Kotsonis, Peter; Frey, Armin; Bömmel, Heike; Schmidt, Harald; Wink, David A.; Pfleiderer, Wolfgang

doi:10.1074/jbc.274.35.24921

Cited by 108 publications

(91 citation statements)

References 58 publications

(88 reference statements)

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“…BH4 appears to facilitate electron transfer from the reductase domain to the oxygenase domain of NOS, and maintains the haem prosthetic group in its redox active form [6,7,11]. Furthermore, BH4 also promotes and possibly stabilises eNOS protein in the active homodimer formation [12][13][14][15][16].…”

Section: Introductionmentioning

confidence: 99%

Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation

et al. 2005

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Aims/hypothesis: Impaired nitric oxide (NO) bioactivity and increased superoxide (SO) production are characteristics of vascular endothelial dysfunction in diabetes. The underlying mechanisms remain unknown. In this regard, we investigated the role of tetrahydrobiopterin (BH4) bioavailability in regulating endothelial nitric oxide synthase (eNOS) activity, dimerisation and SO production in streptozotocin-induced diabetic mice. Methods: Mouse aortas were used for assays of the following: (1) aortic function by isometric tension; (2) NO by electronic paramagnetic resonance; (3) SO by lucigenin-enhanced chemiluminescence and dihydroethidine fluorescence; (4) total biopterin and BH4 by high-performance liquid chromatography; and (5) eNOS protein expression and dimerisation by immunoblotting. Results: In diabetic mouse aortas, relaxations to acetylcholine and NO levels were significantly decreased, but SO production was increased, in association with reductions in total biopterins and BH4. Although total eNOS levels were increased in diabetes, the protein mainly existed in monomeric form. Conversely, specifically augmented BH4 in diabetic endothelium preserved eNOS dimerisation, but the expression remained unchanged. Conclusions/interpretation: Our results demonstrate that BH4 plays an important role in regulating eNOS activity and its functional protein structure, suggesting that increasing endothelial BH4 and/or protecting it from oxidation may be a rational therapeutic strategy to restore eNOS function in diabetes.

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Section: Introductionmentioning

confidence: 99%

Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation

et al. 2005

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“…Native enzyme was purified from pig cerebellum with previously described methods [17,20,45] by DEAE ion-exchange chromatography and 2h,5h-ADP-Sepharose affinity chromatography (yield of 1.0-1.4 mg of protein from 1 kg of tissue, with a specific activity of up to 463 nmol of -citrulline\min per mg). The amount of enzyme-bound H % Bip was determined by reversephase HPLC coupled with fluorimetric detection (λ ex l 352 nm, λ em l 438 nm), with authentic reagent H % Bip as a standard [17,25,42]. Native NOS-I (0.8-1.0 µg) was incubated in 50 mM Tris\HCl buffer, pH 7.2, containing 1 mM CaCl # , 10 µM FAD, 5 µM FMN, 250 µM CHAPS, 1 mM NADPH for 15 min at either 4 or 37 mC.…”

Section: Determination Of Nos-bound H 4 Bipmentioning

confidence: 99%

“…To determine H % Bip, samples were then centrifuged (10 000 g at 4 mC for 20 min) through 10 kDa cut-off filters (Millipore, Eschborn, Germany). The lowmolecular-mass filtrate contained pterin displaced from NOS [17,25,42]. For determinations of enzyme-bound pterin, the NOScontaining residue was redissolved in Tris\HCl buffer, pH 6.7, and immediately oxidized to biopterin after treatment for 1 h in the dark with 0.2 M I # and 0.5 M KI in the presence of either 0.5 M HCl (acidic oxidation) or 0.5 M NaOH (alkaline oxidation).…”

Section: Determination Of Nos-bound H 4 Bipmentioning

confidence: 99%

“…However, the current consensus in the literature does not support such a classical catalytic role [13][14][15][16][17], although some studies can be found to the contrary [18]. Alternatively, protective and stabilizing effects of H % Bip have been described, including (1) the stabilization of the NOS mRNA product [19], (2) protection of the NOS catalytic centre from oxidative damage [16,20], (3) prevention of the formation of superoxide (O # − d) [21] and H # O # [22] by coupling reductive oxygen activation to -arginine oxidation, and (4) promotion of subunit assembly from monomers and stabilization of dimers in the presence of chemical denaturants [23,24] or under native conditions during -arginine turnover [17,25]. Interestingly, the stabilizing effects of H % Bip might be isoform-dependent.…”

Section: Introductionmentioning

confidence: 99%

“…Interestingly, the stabilizing effects of H % Bip might be isoform-dependent. For NOS-I, H % Bip is required for dimer stabilization [17,24,25] but not for subunit assembly [26,27] ; for NOS-II and III, the current evidence that supports a role for H % Bip in subunit assembly is inconsistent [23,[28][29][30][31][32][33] even when the enzymes have been solved at the crystal level [16,34].…”

Section: Introductionmentioning

confidence: 99%

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Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide

Kotsonis

Fröhlich

Shutenko

et al. 2000

Biochemical Journal

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The underlying mechanisms regulating the activity of the family of homodimeric nitric oxide synthases (NOSs) and, in particular, the requirement for (6R)-5,6,7,8-tetrahydro-L-biopterin (H4Bip) are not fully understood. Here we have investigated possible allosteric and stabilizing effects of H4Bip on neuronal NOS (NOS-I) during the conversion of substrate, L-arginine, into L-citrulline and nitric oxide. Indeed, in kinetic studies dual allosteric interactions between L-arginine and H4Bip activated recombinant human NOS-I to increase L-arginine turnover. Consistent with this was the observation that H4Bip, but not the pterin-based NOS inhibitor 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octahydro-oxazolo[1,2-f]-pteridine (PHS-32), caused an L-arginine-dependent increase in the haem Soret band, indicating an increase in substrate binding to recombinant human NOS-I. Conversely, L-arginine was observed to increase in a concentration-dependent manner H4Bip binding to pig brain NOS-I. Secondly, we investigated the stabilization of NOS quaternary structure by H4Bip in relation to uncoupled catalysis. Under catalytic assay conditions and in the absence of H4Bip, dimeric recombinant human NOS-I dissociated into inactive monomers. Monomerization was related to the uncoupling of reductive oxygen activation, because it was inhibited by both superoxide dismutase and the inhibitor NΩ-nitro-L-arginine. Importantly, H4Bip was found to react chemically with superoxide (O2-−) and enzyme-bound H4Bip was consumed under O2-−-generating conditions in the absence of substrate. These results suggest that H4Bip allosterically activates NOS-I and stabilizes quaternary structure by a novel mechanism involving the direct interception of auto-damaging O2-−.

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Inducible Nitric Oxide Synthase

Rosenfeld¹,

Tainer²,

Getzoff³

2004

Handbook of Metalloproteins

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Tetrahydrobiopterin Inhibits Monomerization and Is Consumed during Catalysis in Neuronal NO Synthase

Abstract: The biosynthesis of nitric oxide (NO) is catalyzed by homodimeric NO synthases (NOS).

Cited by 108 publications

References 58 publications

Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation

Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation

Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide

Inducible Nitric Oxide Synthase

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