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 H-3-labeled (6R)-5,6,7,8-tetrahydro-L-biopterin as radioligand. Asso ciation of [H-3]tetrahydrobiopterin followed second order kinetics (k(on) = 1.3 x 10(6) M(-1) min(-1)), the dissociation reaction was reversible and first-order (k(off) = 3.2 x 10(-1) min(-1)), yielding a kinetic K-D of 0.25 mu M. Binding of the radioligand was competitively antagonized by several pteridine derivatives with the following order of potency (K-I): 7,8-dihydro-L-biopterin (2.2 mu M), (6S)-5,6,7,8-tetrahydro- L-biopterin (19 mu M), (6R,S)-6-methyl-5,6,7,8-tetrahydropterin (240 mu M), and 6,7-dimethyl-5,6,7,8-tetrahydropterin (> 1 mM). The affinity of NO synthase for tetrahydrobiopterin was increased 6-fold in the pres ence of 0.1 mM L-argnine (K-D = 37 nM), and, conversely, tetrahydrobiopterin enhanced the affinity of the enzyme for H-3-labeled N-G-nitro-L-arginine about 2-fold. 7-Nitroindazole, which presumably binds to the heme group of NO synthase, competitively inhibited binding of [H-3]tetrahydrobiopterin and [H-3]N-G-nitro-L-arginine with similar K-I values (0.1 mu M). 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.