Structural requirements for human inducible nitric oxide synthase substrates and substrate analogue inhibitors

摘要

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. Desamino-arginine, 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 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 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-H-3]-N-6-(1-iminoethyl)lysine indicates that the mechanism of enzyme inactivation is not covalent modification of the protein. [References: 28]