title: Structural requirements for human inducible nitric oxide synthase substrates and substrate analogue inhibitors.
abstract: 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-N6-(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-N6-(1-iminoethyl)lysine, L-N5-(1-iminoethyl)ornithine, and N5-(1-iminoethyl)cadaverdine, but not N6-(1-iminoethyl)-6-aminocaproic acid, were NADPH-dependent, irreversible inactivators of iNOS. For both the L-Arg and L-N6-(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-N6-(1-iminoethyl)lysine was NADPH- and dioxygen-dependent, but low incorporation of radiolabel with DL--4, 5-3H]-N6-(1-iminoethyl)lysine indicates that the mechanism of enzyme inactivation is not covalent modification of the protein.
authors.affiliation: Department of Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065, USA. stephanvgrant@merck.com
authors.forename: S K
authors.initials: SK
authors.lastname: Grant
authors.forename: B G
authors.initials: BG
authors.lastname: Green
authors.forename: J
authors.initials: J
authors.lastname: Stiffey-Wilusz
authors.forename: P L
authors.initials: PL
authors.lastname: Durette
authors.forename: S K
authors.initials: SK
authors.lastname: Shah
authors.forename: J W
authors.initials: JW
authors.lastname: Kozarich
mesh_terms: D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D006838:Hydrocarbons; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry; D008697:Methane / Q000031:analogs & derivatives / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D004791:Enzyme Inhibitors; D006838:Hydrocarbons; C090663:N(6)-(1-iminoethyl)lysine; C030011:carbene; D001120:Arginine; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D008239:Lysine; D008697:Methane