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.
Klatt
P
P
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität, Graz, Austria.
Schmid
M
M
Leopold
E
E
Schmidt
K
K
Werner
E R
ER
Mayer
B
B
D000494:Allosteric Regulation; D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D007191:Indazoles / Q000494:pharmacology; D007700:Kinetics; D019001:Nitric Oxide Synthase; D011621:Pteridines / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
D007191:Indazoles; D011621:Pteridines; D001708:Biopterins; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; C003402:sapropterin; C080122:7-nitroindazole