The pteridine binding site of brain nitric oxide synthase. Tetrahydrobiopterin binding kinetics, specificity, and allosteric interaction with the substrate domain.
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.[1]References
- The pteridine binding site of brain nitric oxide synthase. Tetrahydrobiopterin binding kinetics, specificity, and allosteric interaction with the substrate domain. Klatt, P., Schmid, M., Leopold, E., Schmidt, K., Werner, E.R., Mayer, B. J. Biol. Chem. (1994) [Pubmed]
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