Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.19 (NOS)
 7,285 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The active site topologies of neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) nitric-oxide synthases heterologously expressed in Escherichia coli have been examined using three aryldiazene (Ar-N=NH) probes. The topological information derives from (a) the rate and extent of aryl-iron complex formation in the presence and absence of tetrahydrobiopterin (H4B), Ca2+-dependent calmodulin (CaM), and L-arginine, and (b) the N-phenylprotoporphyrin IX regioisomer ratios obtained upon migration of the phenyl of the phenyl-iron complex to the heme nitrogen atoms. The N-phenylprotoporphyrin ratios indicate that the three NOS isoforms have related active site topologies with unencumbered space above all four pyrrole rings but particularly above pyrrole ring D. H4B binds directly above the heme pyrrole ring D or causes a conformational change that constricts that region, because H4B markedly decreases phenyl migration to pyrrole ring D. Small CaM-dependent changes in the nNOS N-phenylporphyrin isomer pattern are consistent with a conformational link between the CaM and heme sites in this protein. The ceiling height directly above the heme iron atom differs among the isoforms and is lower than in the P450 enzymes because only nNOS and iNOS react with 2-naphthyldiazene, and none of the isoforms reacts with p-biphenyldiazene. L-Arg blocks access to the heme iron atom in all three NOS isoforms and nearly suppresses the phenyldiazene reaction. The data indicate that topological differences, including differences in the size of the active site, are superimposed on the structural similarities among the NOS active sites.
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PMID:Active site topologies and cofactor-mediated conformational changes of nitric-oxide synthases. 904 46

Neuronal nitric oxide synthase (nNOS) catalyzes the oxidation of NG-hydroxy-L-arginine (NHA) by hydrogen peroxide. The amino acid products were characterized by high-performance liquid chromatography/mass spectrometry of the o-phthalaldehyde/2-mercaptoethanol derivatives and identified as citrulline and N delta-cyanoornithine (CN-orn). The assignment of CN-orn was confirmed by independent chemical synthesis and comparison of the properties of the enzyme-derived product with those of synthetic CN-orn. The inorganic products detected in the enzymatic reaction were NO2- and NO3-, presumably from oxidation of NO-. The reaction of H2O2 and NHA with nNOS was at least 10-fold slower than the reaction of NADPH, O2, and NHA (Vmax,app = 49 +/- 2 nmol min-1 mg-1 for the reactions with 10 microM added H4B). The reaction exhibited saturation kinetics with respect to hydrogen peroxide [K(m,app)(H2O2) = 10 +/- 1 mM for the reactions with 10 microM added H4B]. No H2O2-dependent reaction was observed with L-arginine as the amino acid substrate. The different products for the NADPH- and H2O2-dependent transformations of NHA are of mechanistic significance in the NOS reaction.
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PMID:Formation of N delta-cyanoornithine from NG-hydroxy-L-arginine and hydrogen peroxide by neuronal nitric oxide synthase: implications for mechanism. 939 65

Nitric oxide synthase, NOS (EC.1.14.13.39), was purified from bovine pancreas over 5,500-fold with a 7.6% yield using 30% ammonium sulfate precipitation, and 2',5'-ADP-agarose and calmodulin-agarose affinity chromatography. The purified bovine pancreatic NOS (bpNOS) showed a single band on SDS-PAGE corresponding to an apparent molecular mass of 160 kDa, whereas it was 320 kDa on non-denaturating gel-filtration. This indicated a homodimeric nature of the enzyme. The specific activity of the purified bpNOS was 31.67 nmol L-citrulline fored/mtn/mg protein and apparent K(m) for L-arginine was 15.72 microM. The enzyme activity was dependent on Ca2+ and calmodulin, and to a lesser extent on NADPH, FAD and FMN. H4B was not required as a cofactor for the activity. In an inhibition experiment with L-arginine analogues, NG-nitro-L-arginine (NNA) had the most potent inhibitory effect on bpNOS, and NG, NG'-dimethyl-L-arginine (symmetric; sDMA) did not have any inhibitory effect. Immunohistochemical analysis of the bovine pancreas using brain type NOS antibody (anti-bNOS antibody) revealed that acinar cells showed strong immunoreactivity against the antibody.
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PMID:Nitric oxide synthase from bovine pancreas: purification and characterization. 987 19

Overproduction of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) has been etiologically linked to several inflammatory, immunological, and neurodegenerative diseases. As dimerization of NOS is required for its activity, several dimerization inhibitors, including pyrimidine imidazoles, are being evaluated for therapeutic inhibition of iNOS. However, the precise mechanism of their action is still unclear. Here, we examined the mechanism of iNOS inhibition by a pyrimidine imidazole core compound and its derivative (PID), having low cellular toxicity and high affinity for iNOS, using rapid stopped-flow kinetic, gel filtration, and spectrophotometric analysis. PID bound to iNOS heme to generate an irreversible PID-iNOS monomer complex that could not be converted to active dimers by tetrahydrobiopterin (H4B) and l-arginine (Arg). We utilized the iNOS oxygenase domain (iNOSoxy) and two monomeric mutants whose dimerization could be induced (K82AiNOSoxy) or not induced (D92AiNOSoxy) with H4B to elucidate the kinetics of PID binding to the iNOS monomer and dimer. We observed that the apparent PID affinity for the monomer was 11 times higher than the dimer. PID binding rate was also sensitive to H4B and Arg site occupancy. PID could also interact with nascent iNOS monomers in iNOS-synthesizing RAW cells, to prevent their post-translational dimerization, and it also caused irreversible monomerization of active iNOS dimers thereby accomplishing complete physiological inhibition of iNOS. Thus, our study establishes PID as a versatile iNOS inhibitor and therefore a potential in vivo tool for examining the causal role of iNOS in diseases associated with its overexpression as well as therapeutic control of such diseases.
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PMID:Mechanism of inducible nitric-oxide synthase dimerization inhibition by novel pyrimidine imidazoles. 2369 43