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.

Biliverdin: the blue-green pigment in the bones of the garfish (Belone belone) and eelpout (Zoarces viviparus)

“…This is in accordance with the MS and MS/MS spectra of the reference substance we investigated, biliverdin dimethyl ester. In the MS 3 of the methylated garfish samples, we found m/z 283 as the main fragment; this is identical with the MS 3 identified by Zhu et al 2005 as the non-methylated biliverdin IX a. No meaningful MS 3 could be derived for eelpout, only small amounts of which were available.…”

“…5a, b, d, e). Zhu et al [20] conducted extensive mass spectrographic analyses of biliverdin isomers. Based on their results, the following masses are characteristic of biliverdin: m/z [M ?…”

BYK191023 (2-[2-(4-Methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine) Is an NADPH- and Time-Dependent Irreversible Inhibitor of Inducible Nitric-Oxide Synthase

“…At least three different mechanisms could explain the irreversible inactivation of iNOS: 1) a time-dependent suicide mechanism is involved wherein the inhibitor is metabolized to a reactive intermediate that covalently binds the enzyme; 2) bound inhibitor may decrease stability of the iNOS dimer by steric effect and induce a time-dependent loss of dimerization [the dimer stability order is iNOS Ͻ nNOS Ͻ Ͻ eNOS (Panda et al, 2002) also in accordance with BYK191023 selectivity]; and 3) uncoupled NADPH oxidation in the inhibitor bound iNOS results in the formation of reactive oxygen species at the heme site, and these may cause alteration and/or loss of the porphyrin ring, as reported previously for 1400W and other NOS inhibitors (Demady et al, 2001;Lee et al, 2005;Zhu et al, 2005).…”

“…In the samples, we detected other very low-level 400-nm-absorbing compounds visible only using higher gain in the detector. We attempted to identify possible NADPH-and time-dependent formation of biliverdin, as suggested by the 1400W inactivation of iNOS (Zhu et al, 2005), but differences with the retention time of commercial biliverdin precluded an accurate identification. We then collected HPLC fractions from [ 3 H]BYK191023 inactivated iNOS every 0.5 min, and we detected radioactivity for each fraction by scintillation.…”

Involvement of inducible nitric oxide synthase in hydroxyl radical-mediated lipid peroxidation in streptozotocin-induced diabetes

“…The iNOS inhibitor 1400W was demonstrated to cause irreversible inhibition of the enzyme by inducing extensive chemical modification of the heme site, rendering the enzyme unable to oxygenate L-arginine [45]. The fact that L-arginine abrogated free radical production is consistent with an iNOS-dependent but transition metal-independent route for hydroxyl radical production, which induces lipid peroxidation in the diabetic animals.…”