ALLOSTERIC MODULATION OF RAT-BRAIN NITRIC-OXIDE SYNTHASE BY THE PTERIN-SITE ENZYME-INHIBITOR 4-AMINOTETRAHYDROBIOPTERIN

We investigated the functional and allosteric effects of the 4-amino a nalogue of tetrahydrobiopterin, rahydro-6-(L-erythro-1,2-dihydroxyprop yl)pteridine (4-amino-H(4)biopterin) on pteridine-free rat neuronal ni tric oxide synthase. In the presence of added (6R)-5,6,7,8-tetrahydro- L-erythrobiopterin (H(4)biopterin; 10 mu M), 4-amino-H(4)biopterin com pletely inhibited the conversion of both L-arginine and N-G-hydroxy-L- arginine with half-maximally effective concentrations of 1.1+/-0.09 an d 1.3+/-0.09 mu M, respectively. Inhibition was reversible, as shown b y a time-dependent restoration of citrulline formation upon dilution o f the inhibitor-treated enzyme (t(1/2) = 3.0 min). Binding of 4-amino- H(4)biopterin led to a complete conversion of the haem from low-spin t o high-spin state, and to the formation of stable homodimers which par tially survived electrophoresis under denaturating conditions. These r esults show that oxidation of both L-arginine and N-G-hydroxy-L-argini ne is pteridine-dependent, and that the allosteric effects of H(4)biop terin do not fully explain the essential role of the pteridine cofacto r in nitric oxide biosynthesis.