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 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 microM), 4-amino-H4biopterin completely inhibited the conversion of both L-arginine and NG-hydroxy-L-arginine with half-maximally effective concentrations of 1.1+/-0.09 and 1.3+/-0.09 microM, respectively. Inhibition was reversible, as shown by a time-dependent restoration of citrulline formation upon dilution of the inhibitor-treated enzyme (t1/2=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 NG-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.
Pfeiffer
S
S
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria.
Gorren
A C
AC
Pitters
E
E
Schmidt
K
K
Werner
E R
ER
Mayer
B
B
D000494:Allosteric Regulation; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D001921:Brain / Q000201:enzymology*; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051381:Rats
D004791:Enzyme Inhibitors; C106930:4-amino-tetrahydrobiopterin; D001708:Biopterins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; C003402:sapropterin