Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide.
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)(-.).
Kotsonis
P
P
Department of Pharmacology, Julius-Maximilians University, Versbacher Strasse 9, D-97078 Würzburg, Germany. Peter.Kotsonis@pharma.novartis.com
Fröhlich
L G
LG
Shutenko
Z V
ZV
Horejsi
R
R
Pfleiderer
W
W
Schmidt
H H
HH
D000494:Allosteric Regulation; D001120:Arginine / Q000494:pharmacology; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D002384:Catalysis; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007202:Indicators and Reagents; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D020836:Protein Structure, Quaternary; D013481:Superoxides / Q000037:antagonists & inhibitors* / Q000378:metabolism
D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D013481:Superoxides; D001708:Biopterins; D001120:Arginine; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C003402:sapropterin