INDUCIBLE NITRIC-OXIDE SYNTHASE - IDENTIFICATION OF AMINO-ACID-RESIDUES ESSENTIAL FOR DIMERIZATION AND BINDING OF TETRAHYDROBIOPTERIN

Nitric oxide synthases (NOSs) require tetrahydrobiopterin (BH4) for di merization and NO production. Mutation analysis of mouse inducible NOS (iNOS; NOS2) identified Gly-450 and Ala-453 as critical for NO produc tion, dimer formation, and BH4 binding. Substitutions at five neighbor ing positions were tolerated, and normal binding of heme, calmodulin, and NADPH militated against major distortions affecting the NH2-termin al portion, midzone, or COOH terminus of the inactive mutants. Direct involvement of residues 450 and 453 in the binding of BH4 is supported by the striking homology of residues 448-480 to a region extensively shared by the three BH4-utilizing aromatic amino acid hydroxylases and is consistent with the conservation of these residues among all 10 re ported NOS sequences, including mammalian NOSs 1, 2, and 3, as well as avian and insect NOSs. Altered binding of BH4 and/or L-arginine may e xplain how the addition of a single methyl group to the side chain of residue 350 or the addition of three methylenes to residue 453 can eac h abolish an enzymatic activity that reflects the concerted function o f 1143 other residues.