title: Mechanism of inactivation of inducible nitric oxide synthase by amidines. Irreversible enzyme inactivation without inactivator modification.
abstract: Nitric oxide synthases (NOS) are hemoproteins that catalyze the reaction of L-arginine to L-citrulline and nitric oxide. N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was reported to be a slow, tight-binding, and highly selective inhibitor of iNOS in vitro and in vivo. Previous mechanistic studies reported that 1400W was recovered quantitatively after iNOS fully lost its activity and modification to iNOS was not detected. Here, it is shown that 1400W is a time-, concentration-, and NADPH-dependent irreversible inactivator of iNOS. HPLC-electrospray mass spectrometric analysis of the incubation mixture of iNOS with 1400W shows both loss of heme cofactor and formation of biliverdin, as was previously observed for iNOS inactivation by another amidine-containing compound, N5-(1-iminoethyl)-L-ornithine (L-NIO). The amount of biliverdin produced corresponds to the amount of heme lost by 1400W inactivation of iNOS. A convenient MS/MS-HPLC methodology was developed to identify the trace amount of biliverdin produced by inactivation of iNOS with either 1400W or L-NIO to be biliverdin IXalpha out of the four possible regioisomers. Two mechanisms were previously proposed for iNOS inactivation by L-NIO: (1) uncoupling of the heme peroxide intermediate, leading to destruction of the heme to biliverdin; (2) abstraction of a hydrogen atom from the amidine methyl group followed by attachment to the heme cofactor, which causes the enzyme to catalyze the heme oxygenase reaction. The second mechanistic proposal was ruled out by inactivation of iNOS with d3-1400W, which produced no d2-1400W. Detection of carbon monoxide as one of the heme-degradation products further excludes the covalent heme adduct mechanism. On the basis of these results, a third mechanism is proposed in which the amidine inactivators of iNOS bind as does substrate L-arginine, but because of the amidine methyl group, the heme peroxy intermediate cannot be protonated, thereby preventing its conversion to the heme oxo intermediate. This leads to a change in the enzyme mechanism to one that resembles that of heme oxygenase, an enzyme known to convert heme to biliverdin IXalpha. This appears to be the first example of a compound that causes irreversible inactivation of an enzyme without itself becoming modified in any way.
authors.affiliation: Department of Chemistry, Drug Discovery Program, Northwestern University, Evanston, Illinois 60208-3113, USA.
authors.forename: Yaoqiu
authors.initials: Y
authors.lastname: Zhu
authors.forename: Dejan
authors.initials: D
authors.lastname: Nikolic
authors.forename: Richard B
authors.initials: RB
authors.lastname: Van Breemen
authors.forename: Richard B
authors.initials: RB
authors.lastname: Silverman
mesh_terms: D000578:Amidines / Q000494:pharmacology*; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology*; D001664:Biliverdine / Q000096:biosynthesis; D002248:Carbon Monoxide / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006418:Heme / Q000378:metabolism; D006736:Horses; D006801:Humans; D007700:Kinetics; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009952:Ornithine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000578:Amidines; D001596:Benzylamines; D004791:Enzyme Inhibitors; C104820:N-(3-(aminomethyl)benzyl)acetamidine; C065027:N(G)-iminoethylornithine; D006418:Heme; D002248:Carbon Monoxide; D009952:Ornithine; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D001664:Biliverdine