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Cited by 219Nitric Oxide and Endothelin EffectsAmino Acid Enzymes and MetabolismHemoglobin structure and function

The pteridine binding site of brain nitric oxide synthase. Tetrahydrobiopterin binding kinetics, specificity, and allosteric interaction with the substrate domain.

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Klatt, P., Schmid, M., Leopold, E., Schmidt, K., Werner, E.R., Mayer, B.·1994·Journal of Biological Chemistry
DOI 10.1016/s0021-9258(17)36726-1
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Abstract

Nitric oxide (NO) synthases contain FAD, FMN, heme, and (6R)-5,6,7,8-tetrahydro-L-biopterin as prosthetic groups. We have characterized the pteridine-binding site of purified brain NO synthase, using 3H-labeled (6R)-5,6,7,8-tetrahydro-L-biopterin as radioligand. Association of [3H]tetrahydrobiopterin followed second-order kinetics (kon = 1.3 x 10(6) M-1 min-1), the dissociation reaction was reversible and first-order (koff = 3.2 x 10(-1) min-1), yielding a kinetic KD of 0.25 microM. Binding of the radioligand was competitively antagonized by several pteridine derivatives with the following order of potency (KI): 7,8-dihydro-L-biopterin (2.2 microM), (6S)-5,6,7,8-tetrahydro-L-biopterin (19 microM), (6R,S)-6-methyl-5,6,7,8-tetrahydropterin (240 microM), and 6,7-dimethyl-5,6,7,8-tetrahydropterin (> 1 mM). The affinity of NO synthase for tetrahydrobiopterin was increased 6-fold in the presence of 0.1 mM L-arginine (KD = 37 nM), and, conversely, tetrahydrobiopterin enhanced the affinity of the enzyme for 3H-labeled NG-nitro-L-arginine about 2-fold. 7-Nitroindazole, which presumably binds to the heme group of NO synthase, competitively inhibited binding of [3H]tetrahydrobiopterin and [3H]NG-nitro-L-arginine with similar Ki values (0.1 microM). Functional as well as binding studies revealed that 7-nitroindazole was competitive with both L-arginine and tetrahydrobiopterin. Our data indicate that brain NO synthase exhibits a highly specific binding site for (6R)-5,6,7,8-tetrahydro-L-biopterin, which allosterically interacts with the substrate domain and may be located proximal to the prosthetic heme group of NO synthase.

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  • l-Arginine is important for the dimerization of NOS and increases BH4 binding affinity.

    “l-Arginine is also important for the dimerization of NOS (51, 515) and increases BH4 binding affinity (514, 753).”

    From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [514]

  • iNOS and nNOS show higher affinity toward BH4 than for BH2 (around 10-fold in the absence of l-arginine).

    “iNOS and nNOS show higher affinity toward BH4 than for BH2 (around 10-fold in the absence of l-arginine) (6, 514, 753); however, eNOS shows almost identical binding affinities for BH4 and BH2, and it is thus very sensitive to imbalances in the concentrations of BH4 and BH2 (190).”

    From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [514]

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Authors
Klatt, P., Schmid, M., Leopold, E., Schmidt, K., Werner, E. R., & Mayer, B.
Affiliations
University of Graz (AT)
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
1994-05
Volume
269
Issue
19
Pages
13861-13866
ISSN
0021-9258 (ISSN-L)
DOI
10.1016/s0021-9258(17)36726-1
OpenAlex
W1499812702
Cited by
219
References
44
Citations by year
2026: 2 · 2025: 1 · 2024: 2 · 2021: 1 · 2020: 1 · 2019: 1 · 2018: 1 · 2017: 4 · 2016: 1 · 2015: 3 · 2014: 4 · 2013: 2 · 2012: 5
FWCI
15.84
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Amino Acid Enzymes and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Hemoglobin structure and function
Keywords
tetrahydrobiopterin, allosteric interactions, binding kinetics, competitive inhibition, 7-nitroindazole, L-arginine, L-NNA, enzyme regulation, prosthetic groups, heme, FAD, flavin mononucleotide
MeSH terms
Allosteric Regulation, Amino Acid Oxidoreductases, Animals, Biopterins, Brain, Indazoles, Kinetics, Pteridines, Substrate Specificity, Swine, Nitric Oxide Synthase

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