title: Mechanistic studies of inactivation of inducible nitric oxide synthase by amidines.
abstract: Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide. N(5)-(1-Iminoethyl)-L-ornithine (L-NIO), an amidine-containing molecule, is a natural product known to be an inactivator of inducible NOS (iNOS). Because of the presence of the amidine methyl group in place of the guanidine amino group of substrate L-arginine, the active site heme peroxy intermediate sometimes cannot be protonated, thereby preventing its conversion to the heme oxo intermediate; instead, a heme oxygenase-type mechanism occurs, leading to conversion of the heme to biliverdin. This might be a new and general inactivation mechanism for heme-containing enzymes. In the studies described here, we attempted to provide support for amidines as substrates and inactivators of iNOS by the design and synthesis of amidine analogues of L-NIO having groups other than the amidine methyl group. No nitric oxide- or enzyme-catalyzed products could be detected by incubation of these amidines with iNOS. Although none of the L-NIO analogues acted as substrates, they all inhibited iNOS; increased inhibitory potency correlated with decreased substituent size. Computer modeling and molecular dynamics simulations were run on 10 and 11 to rationalize why these compounds do not act as substrates. Unlike the methyl amidine (L-NIO), the other alkyl groups block binding of O2 at the heme iron. Compounds 8, 9, and 11 were inactivators; however, no heme was lost, and no biliverdin was formed. No kinetic isotope effect on inactivation was observed with perdeuterated ethyl 8. A small amount of dimer disruption occurred with these inactivators, although the amount would not account for complete enzyme inactivation. The L-NIO analogues inactivate iNOS by a yet unknown mechanism; however, it is different from that of L-NIO, and the inactivation mechanism previously reported for L-NIO appears to be unique to methyl amidines.
authors: †Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, and Center for Molecular Innovation and Drug Discovery, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States.
authors: Wei
authors: W
authors: Tang
authors: ‡Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, California 92697-3900, United States.
authors: Huiying
authors: H
authors: Li
authors: ‡Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, California 92697-3900, United States.
authors: Thomas L
authors: TL
authors: Poulos
authors: †Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, and Center for Molecular Innovation and Drug Discovery, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States.
authors: Richard B
authors: RB
authors: Silverman
mesh_terms: D000578:Amidines / Q000737:chemistry*; D000818:Animals; D002384:Catalysis; D003198:Computer Simulation; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D009952:Ornithine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D000578:Amidines; D004791:Enzyme Inhibitors; C065027:N(G)-iminoethylornithine; D006418:Heme; D009952:Ornithine; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse