Allosteric modulation of rat brain nitric oxide synthase by the pterin-site enzyme inhibitor 4-aminotetrahydrobiopterin

Abstract

We investigated the functional and allosteric effects of the 4-amino analogue of tetrahydrobiopterin, (6R)-2,4-diamino-5,6,7,8-tetrahydro-6-(l-erythro-1,2-dihydroxypropyl)pteridine (4-amino-H₄biopterin) on pteridine-free rat neuronal nitric oxide synthase. In the presence of added (6R)-5,6,7,8-tetrahydro-L-erythrobiopterin (H₄biopterin; 10 μM), 4-amino-H₄biopterin completely inhibited the conversion of both L-arginine and N^G-hydroxy-L-arginine with half-maximally effective concentrations of 1.1±0.09 and 1.3±0.09 μM, respectively. Inhibition was reversible, as shown by a time-dependent restoration of citrulline formation upon dilution of the inhibitor-treated enzyme (t_1/2 = 3.0 min). Binding of 4-amino-H₄biopterin led to a complete conversion of the haem from low-spin to high-spin state, and to the formation of stable homodimers which partially survived electrophoresis under denaturating conditions. These results show that oxidation of both L-arginine and N^G-hydroxy-L-arginine is pteridine-dependent, and that the allosteric effects of H₄biopterin do not fully explain the essential role of the pteridine cofactor in nitric oxide biosynthesis.