STRUCTURAL REQUIREMENTS FOR HUMAN INDUCIBLE NITRIC-OXIDE SYNTHASE SUBSTRATES AND SUBSTRATE-ANALOG INHIBITORS

Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) catalyzes the NA DPH-dependent oxidation of one of the free guanidino nitrogens of L-Ar g to form nitric oxide and L-citrulline. Analogues of L-Arg and the in hibitor, L-N-6-(1-iminoethyl)lysine, were used to define structural el ements required for the binding and catalysis of compounds. L-Arg anal ogues with sequentially shorter methylene spacing between the guanidin o group and the amino acid portion of the molecule were not iNOS subst rates but were reversible inhibitors. L-Arg analogues such as agmatine with a hydroxyl substitution at the 2-amino position were substrates. Desaminoarginine was not a substrate but a reversible inhibitor. Desa mino-arginine, agmatine, and argininic acid bound to the enzyme to giv e type I difference spectra similar to that of L-Arg. The amidino comp ounds L-N-6-(1-iminoethyl)lysine, L-N-5-(1-iminoethyl)ornithine, and N -5-( 1-iminoethyl)cadaverdine, but not N-6-(1-iminoethyl)-6-aminocapro ic acid, were NP?DPH-dependent, irreversible inactivators of iNOS. For both the L-Arg and L-N-6-(1-iminoethyl)lysine analogues, the 2-amino group appeared to play an important role in catalytic events leading t o either substrate turnover or mechanism-based inactivation. Inactivat ion of iNOS by L-N-6-(1-iminoethyl)lysine was NADPH-and dioxygen-depen dent, but low incorporation of radiolabel with DL-[4,5-H-3]-N-6-(1-imi noethyl)lysine indicates that the mechanism of enzyme inactivation is not covalent modification of the protein.