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| MULTIPLE CATALYTIC FUNCTIONS OF BRAIN NITRIC-OXIDE SYNTHASE - BIOCHEMICAL-CHARACTERIZATION, COFACTOR-REQUIREMENT, AND THE ROLE OF N(G.W)-HYDROXY-L-ARGININE AS AN INTERMEDIATE |
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| Author(s): KLATT P, SCHMIDT K, URAY G, MAYER B |
| Source: JOURNAL OF BIOLOGICAL CHEMISTRY Volume: 268 Issue: 20 Pages: 14781-14787 Published: JUL 15 1993 |
| Times Cited: 238 References: 56 |
| Abstract: Brain NO (nitric oxide) synthase contains FAD, FMN, heme, and tetrahydrobiopterin as prosthetic groups and represents a multi-functional oxidoreductase catalyzing oxidation of L-arginine to NO and L-citrulline, formation of H2O2, and reduction of cytochrome c. We show that substrate analogues and inhibitors interacting with the heme block both the reductive activation of oxygen and the oxidation of L-arginine without affecting cytochrome c reduction. We further demonstrate that N(omega)-hydroxy-L-arginine is an intermediate in enzymatic NO synthesis. The ratio of L-citrulline to free N(omega)-hydroxy-L-arginine was greater-than-or-equal-to 50 under various assay conditions, but could markedly be reduced down to 4 by redox active inhibitors. Brain NO synthase is shown to utilize both L-arginine and N(omega)-hydroxy-L-arginine for the formation of stoichiometric amounts of NO and L-citrulline. Tetrahydrobiopterin equally enhanced reaction rates from either substrate (approximately 5-fold), but its rate accelerating effects were only observed at NADPH concentrations greater-than-or-equal-to 3 muM. In the absence of L-arginine or tetrahydrobiopterin, brain NO synthase catalyzes the generation of H2O2. We now show that, in contrast to L-arginine, N(omega)-hydroxy-L-arginine fully blocked H2O2 formation in the absence of exogenous tetrahydrobiopterin, indicating that N(omega)-hydroxy-L-arginine is a direct inhibitor of enzymatic oxygen activation. Based on these data, a hypothetical mechanism of enzymatic NO formation is discussed. |
| Document Type: Article |
| Language: English |
Addresses:
1. GRAZ UNIV, INST PHARMACOL & TOXIKOL, UNIV PL 2, A-8010 GRAZ, AUSTRIA
2. GRAZ UNIV, INST ORGAN CHEM, A-8010 GRAZ, AUSTRIA |
| Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: LL759 |
| ISSN: 0021-9258 |
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| | | | | | | | | Record from Web of Science® | | | | | | | | | |