Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Kotsonis, P; Fröhlich, LG; Shutenko, ZV; Horejsi, R; Pfleiderer, W; Schmidt, HH.
Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide.
Biochem J. 2000; 346 Pt 3(2):767-776
Doi: 10.1042%2F0264-6021%3A3460767
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- Co-Autor*innen der Med Uni Graz
- Horejsi Renate
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- Abstract:
- 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 (H(4)Bip) are not fully understood. Here we have investigated possible allosteric and stabilizing effects of H(4)Bip 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 H(4)Bip activated recombinant human NOS-I to increase L-arginine turnover. Consistent with this was the observation that H(4)Bip, but not the pterin-based NOS inhibitor 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octahydrooxazolo[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 H(4)Bip binding to pig brain NOS-I. Secondly, we investigated the stabilization of NOS quaternary structure by H(4)Bip in relation to uncoupled catalysis. Under catalytic assay conditions and in the absence of H(4)Bip, 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 N(omega)-nitro-L-arginine. Importantly, H(4)Bip was found to react chemically with superoxide (O(2)(-.)) and enzyme-bound H(4)Bip was consumed under O(2)(-.)-generating conditions in the absence of substrate. These results suggest that H(4)Bip allosterically activates NOS-I and stabilizes quaternary structure by a novel mechanism involving the direct interception of auto-damaging O(2)(-.).
- Find related publications in this database (using NLM MeSH Indexing)
- Allosteric Regulation -
- Arginine - pharmacology
- Binding Sites - pharmacology
- Biopterin - analogs and derivatives
- Catalysis - analogs and derivatives
- Cell Line - analogs and derivatives
- Enzyme Activation - analogs and derivatives
- Enzyme Inhibitors - metabolism
- Humans - metabolism
- Indicators and Reagents - metabolism
- Nitric Oxide Synthase - antagonists and inhibitors
- Nitric Oxide Synthase Type I - antagonists and inhibitors
- Protein Structure, Quaternary - antagonists and inhibitors
- Superoxides - antagonists and inhibitors
- Find related publications in this database (Keywords)
- H4Bip modulation
- monomerization
- nitric oxide synthase catalysis
- reactive oxygen species