Tetrahydrobiopterin-Free Neuronal Nitric Oxide Synthase:  Evidence for Two Identical Highly Anticooperative Pteridine Binding Sites

Gorren, Antonius C.F.; List, Barbara M.; Schrammel, Astrid; Pitters, Eva; Hemmens, Benjamin; Werner, Ernst R.; Schmidt, Kurt; Mayer, Bernd

doi:10.1021/bi961931j

Cited by 147 publications

(177 citation statements)

References 45 publications

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“…In contrast, the full inhibition of basal activity by the type II anti-pterins was not due to loss of endogenous H 4 Bip from NOS (90 Ϯ 7% of control, 100 M PHS-72). It is possible that type II anti-pterins can only bind to the unoccupied, low affinity pterin-binding site of the NOS dimer (37). Nevertheless, inhibition by PHS-72 was fully reversible and inhibition was partially competitive with respect to H 4 Bip (Fig.…”

Section: Anti-pterins a Novel Class Of Pterinmentioning

confidence: 97%

Anti-pterins as Tools to Characterize the Function of Tetrahydrobiopterin in NO Synthase

Bömmel

Reif²,

Fröhlich³

et al. 1998

Journal of Biological Chemistry

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Nitric oxide synthases (NOS) are homodimeric enzymes that NADPH-dependently convert L-arginine to nitric oxide and L-citrulline. Interestingly, all NOS also require (6R)-5,6,7,8-tetrahydro-L-biopterin (H 4 Bip) for maximal activity although the mechanism is not fully understood. Basal NOS activity, i.e. that in the absence of exogenous H 4 Bip, has been attributed to enzyme-associated H 4 Bip. To elucidate further H 4 Bip function in purified NOS, we developed two types of pterin-based NOS inhibitors, termed anti-pterins. In contrast to type II anti-pterins, type I anti-pterins specifically displaced enzyme-associated H 4 Bip and inhibited H 4 Bip-stimulated NOS activity in a fully competitive manner but, surprisingly, had no effect on basal NOS activity. Moreover, for a number of different NOS preparations basal activity (percent of V max ) was frequently higher than the percentage of pterin saturation and was not affected by preincubation of enzyme with H 4 Bip. Thus, basal NOS activity appeared to be independent of enzyme-associated H 4 Bip. The lack of intrinsic 4a-pterincarbinolamine dehydratase activity argued against classical H 4 Bip redox cycling in NOS. Rather, H 4 Bip was required for both maximal activity and stability of NOS by binding to the oxygenase/dimerization domain and preventing monomerization and inactivation during L-arginine turnover. Since anti-pterins were also effective in intact cells, they may become useful in modulating states of pathologically high nitric oxide formation.

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Section: Anti-pterins a Novel Class Of Pterinmentioning

confidence: 97%

Anti-pterins as Tools to Characterize the Function of Tetrahydrobiopterin in NO Synthase

Bömmel

Reif²,

Fröhlich³

et al. 1998

Journal of Biological Chemistry

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“…The reasons for this discrepancy are unclear but might reflect isoform differences or, alternatively, methodological differences, e.g. the use of H % Bip-free NOS and measurements under catalytic [40] compared with non-catalytic conditions used in radioligand binding assays [35][36][37][38][39]. There is therefore uncertainty about the functional significance of allosteric binding site interactions in regulating NOS activity and NO generation.…”

Section: Discussionmentioning

confidence: 99%

“…Allosteric modulation within the catalytic centre of NOS by H % Bip has also been reported [35][36][37][38][39][40]. In a radioligand binding study, Mayer and co-workers demonstrated a 6-fold increase in the affinity of NOS-I for [$H]H % Bip in the presence of -arginine [35].…”

Section: Introductionmentioning

confidence: 98%

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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“…Recent studies revealed that, in addition to redox regulation, H R B also regulates the homodimeric conformation of all three isoforms of NOS. Each nNOS dimer has two identical, anticooperative, binding sites for both Arg and H R B [6]. Incubation with saturating concentrations of H R B induces substantial conformational changes in the homodimeric structure of nNOS, yielding a stabilized nNOS dimer with full NO-producing activity [6,7].…”

Section: Introductionmentioning

confidence: 99%

“…Each nNOS dimer has two identical, anticooperative, binding sites for both Arg and H R B [6]. Incubation with saturating concentrations of H R B induces substantial conformational changes in the homodimeric structure of nNOS, yielding a stabilized nNOS dimer with full NO-producing activity [6,7]. Stabilized nNOS dimer is resistant to 2% sodium dodecyl sulfate (SDS) at temperatures below 30³C, enabling separation of stabilized and non-stabilized nNOS dimers by means of low-temperature SDS-polyacrylamide gel electrophoresis (LT-SDS-PAGE) [7].…”

Section: Introductionmentioning

confidence: 99%

Tetrahydrobiopterin‐dependent stabilization of neuronal nitric oxide synthase dimer reduces susceptibility to phosphorylation by protein kinase C in vitro

Okada

1998

FEBS Letters

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Binding of (6R)-5,6,7,8-tetrahydro-L-biopterin (H 4 B) stabilizes the homodimeric structure of neuronal nitric oxide synthase (nNOS). In the present study, low-temperature sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed differential susceptibility of stabilized and non-stabilized dimers to in vitro phosphorylation by protein kinase C. Protein kinase C preferentially phosphorylated the non-stabilized dimer. Although a low extent of phosphorylation was detected in the stabilized dimer, most of it was estimated to be due to phosphorylation of the dimer before its stabilization. Phosphorylation did not affect the stabilizing effect of H 4 B. These results indicate that H 4 Bdependent dimer stabilization prevents nNOS from protein kinase C-dependent phosphorylation in vitro.z 1998 Federation of European Biochemical Societies.

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Tetrahydrobiopterin-Free Neuronal Nitric Oxide Synthase: Evidence for Two Identical Highly Anticooperative Pteridine Binding Sites

Cited by 147 publications

References 45 publications

Anti-pterins as Tools to Characterize the Function of Tetrahydrobiopterin in NO Synthase

Anti-pterins as Tools to Characterize the Function of Tetrahydrobiopterin in NO Synthase

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

Tetrahydrobiopterin‐dependent stabilization of neuronal nitric oxide synthase dimer reduces susceptibility to phosphorylation by protein kinase C in vitro

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