Structural Requirements for Human Inducible Nitric Oxide Synthase Substrates and Substrate Analogue Inhibitors

Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) catalyzes the NADPH-dependent oxidation of one of the free guanidino nitrogens of l-Arg to form nitric oxide and l-citrulline. Analogues of l-Arg and the inhibitor, l-N 6-(1-iminoethyl)lysine, were used to define structural elements required for the binding and catalysis of compounds. l-Arg analogues with sequentially shorter methylene spacing between the guanidino group and the amino acid portion of the molecule were not iNOS substrates 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. Desaminoarginine, agmatine, and argininic acid bound to the enzyme to give type I difference spectra similar to that of l-Arg. The amidino compounds 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-aminocaproic acid, were NADPH-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 to either substrate turnover or mechanism-based inactivation. Inactivation of iNOS by l-N 6-(1-iminoethyl)lysine was NADPH- and dioxygen-dependent, but low incorporation of radiolabel with dl-[4,5-3H]-N 6-(1-iminoethyl)lysine indicates that the mechanism of enzyme inactivation is not covalent modification of the protein.