From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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ABSTRACT |
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Nitric oxide synthase (NOS) catalyzes the NADPH-
and O2-dependent conversion of
L-arginine to nitric oxide (NO) and citrulline; three
isoforms, the neuronal (nNOS), endothelial, and inducible, have been
identified. Because overproduction of NO is known to contribute to
several pathophysiological conditions, NOS inhibitors are of interest
as potential therapeutic agents. Inhibitors that are potent,
mechanism-based, and relatively selective for the NOS isoform causing
pathology are of particular interest. In the present studies we
report that vinyl-L-NIO
(N5-(1-imino-3-butenyl)-L-ornithine;
L-VNIO) binds to and inhibits nNOS in competition
with L-arginine (Ki = 100 nM); binding is accompanied by a type I optical difference
spectrum consistent with binding near the heme cofactor without
interaction as a sixth axial heme ligand. Such binding is fully
reversible. However, in the presence of NADPH and O2,
L-VNIO irreversibly inactivates nNOS
(kinact = 0.078 min1;
KI = 90 nM); inactivation is
Ca2+/calmodulin-dependent. The cytochrome
c reduction activity of the enzyme is not affected by such
treatment, but the L-arginine-independent NADPH oxidase
activity of nNOS is lost in parallel with the overall activity.
Spectral analyses establish that the nNOS heme cofactor is lost or
modified by L-VNIO-mediated mechanism-based inactivation of
the enzyme. The inducible isoform of NOS is not inactivated by
L-VNIO, and the endothelial isoform requires 20-fold higher concentrations to attain ~75% of the rate of inactivation seen with
nNOS. Among the NOS inactivating L-arginine derivatives, L-VNIO is the most potent and nNOS-selective reported to
date.
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INTRODUCTION |
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Nitric oxide synthase
(NOS)1 catalyzes the
oxidation of L-arginine to nitric oxide (NO) and
citrulline; NADPH and O2 are cosubstrates (1-3). Three
major isoforms of NOS have been identified to date. The neuronal (nNOS)
and endothelial (eNOS) isoforms are constitutively expressed and are
regulated by Ca2+/calmodulin, whereas the activity of the
inducible isoform (iNOS) is controlled transcriptionally and is not
affected by changes in intracellular Ca2+. Although amino
acid sequence homology among the isoforms is limited (~50%) (3), all
are comprised of a C-terminal reductase domain that binds NADPH and the
cofactors FAD and FMN and a N-terminal oxygenase domain that binds
L-arginine and the heme and tetrahydrobiopterin (BH4) cofactors (1-3). The reductase domain is related in
function and amino acid sequence to cytochrome P450 reductase (4),
whereas the oxygenase domain is related in function (but not sequence) to the cytochromes P450. Binding of Ca2+/calmodulin to a
region between the domains permits electron flow from the reductase
domain to the oxygenase domain and also stimulates electron flow within
the reductase domain (5). The resulting reduction of the heme cofactor
allows O2 to be activated, permitting the cytochrome
P450-like oxidation of L-arginine to
N-hydroxy-L-arginine and
the subsequent further oxidation of that tightly bound intermediate to
citrulline and NO (1, 2, 6).
Nitric oxide synthase-derived NO is important in many physiological processes including blood pressure homeostasis (7-9), neurotransmission (10, 11), and immune function (12), but overproduction of NO can have pathological consequences (13). For example, excess NO resulting from overexpression of iNOS in response to endotoxin or inflammatory cytokines is a major contributor to the vascular disregulation seen in septic shock (14, 15) and in patients receiving interleukin-2-based immunotherapy (16, 17). Inappropriate activation of nNOS is implicated in chronic visceral pain (18, 19), in migraine headache (20), and in several neurodegenerative diseases (e.g. Parkinson's disease (21, 22)), and is thought to contribute to post-ischemic reperfusion injury in stroke (23).
Appreciation of the pathological roles of NOS-derived NO has stimulated
interest in the design and synthesis of NOS inhibitors for possible
therapeutic use in disorders associated with overproduction of NO (24,
25). To be pharmacologically useful, compounds should be well
transported into the target tissue, strongly inhibitory, NOS
isoform-selective, and chemically stable under biological conditions.
In attempting to meet these goals, L-arginine analogs are
particularly attractive inhibitor candidates because they are
effectively transported by the ubiquitous system y+ amino
acid transporter (26, 27) and thus show good activity in
vivo.
N-Methyl-L-arginine
(L-NMA), the prototypic NOS inhibitor (28), has been shown,
for example, to reverse the hypotension of septic shock (14, 15, 29)
and cytokine-induced shock (17, 30, 31) in animal studies and in early
clinical trials. Unfortunately, NMA and several other
L-arginine analogs including
N
-amino-L-arginine
(32-34) and
N
-(1-iminoethyl)-L-ornithine
(L-NIO) (35, 36) show little isoform selectivity; their
pharmacological use may thus cause undesirable inhibition of
physiological processes controlled by nontargeted NOS isoforms. We (37,
38) and others (39) have shown that the
S-alkyl-L-thiocitrullines show modest
selectivity (up to 50-fold) for nNOS over eNOS and iNOS and have
proposed that these compounds may be of use in treating disorders
involving overstimulation of nNOS (e.g. stroke). Improved
potency and isoform selectivity would, however, be highly
desirable.
In the present studies, we have examined several novel L-arginine antagonists and find that vinyl-L-NIO (N5-(1-imino-3-butenyl)-L-ornithine, L-VNIO) (Fig. 1) is a potent, mechanism-based inhibitor that attacks the heme cofactor of NOS. It shows a marked selectivity for nNOS. Abstracts describing this work have been published (40, 41).
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EXPERIMENTAL PROCEDURES |
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Materials
Most biochemicals and reagents for organic syntheses were
obtained from Sigma and Aldrich, respectively.
N-Methyl-L-arginine and
BH4 were purchased from Chemical Dynamics (Plainfield, NJ)
and Alexis (La Jolla, CA), respectively.
L-[14C]Arginine was from NEN Life Science
Products.
N
-Alkyl-L-arginines (42)
and L-NIO (43) were prepared by the general methods
indicated. Rat nNOS was isolated from stably transfected kidney 293 cells (44) as described previously (45). Bovine eNOS (46) and mouse
iNOS expressed in Escherichia
coli2 were generous
gifts from Dr. Kirkwood Pritchard (Department of Pathology, Medical
College of Wisconsin, Milwaukee, WI) and Dr. Bettie S. S. Masters
(Department of Biochemistry, University of Texas Health Sciences
Center, San Antonio, TX), respectively. 1H and
13C NMR spectra were obtained using a Bruker AC 300 MHz
spectrometer. FAB mass spectral analyses were generously carried out by
Dr. Frank Laib at the Department of Chemistry, University of Wisconsin, Milwaukee.
General Procedure for Preparation of N5-(1-Iminoalkyl)ornithines and N5-(1-Iminoalkenyl)ornithines
D- or L-Ornithine HCl (1.68 g, 10 mmol) and cupric acetate (0.5 g, 10 mmol) were dissolved in water (20 ml) and stirred for 10 min at room temperature. The solution was then filtered to remove minor impurities and cooled to 0 °C, and the pH was adjusted to 9.5 by addition of cold 10% NaOH. Alkyl or alkenyl imidate (15 mmol), prepared separately from the corresponding nitrile and HCl(g) (43) was then added, and the mixture was allowed to stir at pH 9.0-9.5 for 1 h at 0 °C and for 2 h at room temperature. The pH was then adjusted to 7.4 with cold dilute HCl, and the mixture was stirred at room temperature overnight. Hydrogen sulfide gas (caution: toxic) was bubbled through the solution, and the resulting copper sulfide precipitate was removed by filtration through charcoal. The filtrate was passed through Chelex to remove any residual Cu2+, and the clear solution was evaporated to dryness by rotary evaporation under reduced pressure. The residue was washed with ethyl acetate, and the product was crystallized from ethanol to give pure N5-(1-iminoalkyl)ornithine or N5-(1-iminoalkenyl)ornithine.
N5-(1-Imino-3-butenyl)-L-ornithine
(Vinyl-L-NIO, L-VNIO)--
m.p. 162 °C
(dec); 1H NMR (D2O): 1.65-2.1 (m, 4H), 3.3 (d, 2H), 3.4(t, 2H), 3.8 (t, 1H), 5.4 (m, 2H), and 5.95 (m, 1H);
13C NMR (D2O):
25.43, 30.35, 39.37, 44.28, 56.93, 124.16, 131.55, 168.72, and 176.98; FABMS: m/e 200 (M + H).
N5-(1-Imino-3-butenyl)-D-ornithine
(Vinyl-D-NIO; D-VNIO)--
m.p. 170 °C
(dec), 1H NMR (D2O): 1.6-2.1 (m, 4H), 3.3 (d, 2H), 3.4(t, 2H), 3.8 (t, 1H), 5.4 (m, 2H), and 5.89 (m, 1H);
13C NMR (D2O):
25.43, 30.35, 39.37, 44.27, 56.93, 124.13, 131.55, 168.72, and 176.98; FABMS: m/e 200 (M + H).
N5-(1-Iminopropyl)-L-ornithine
(Methyl-L-NIO)--
m.p. 150 °C. (dec), 1H
NMR (D2O): 1.29 (t, 3H), 1.6-1.9 (m, 4H), 2.54 (q,
2H), 3.38 (t, 2H), and 3.84 (t, 1H); 13C NMR
(D2O):
13.37, 25.43, 28.10, 30.35, 44.09, 56.94, 171.97, and 177.03; FABMS: m/e 188 (M + H).
N5-(1-Iminobutyl)-L-ornithine
(Ethyl-L-NIO)--
m.p. 145 °C (dec), 1H
NMR (D2O): 1.01 (t, 3H), 1.65-2.1 (m, 6H), 2.5 (t,
2H), 3.4(t, 2H), and 3.8 (t, 1H); 13C NMR
(D2O):
15.06, 22.55, 25.50, 30.44, 37.16, 44.12, 56.97, 170.78, and 177.02; FABMS: m/e 202 (M + H).
Methods
Ki Determination-- Activity of NOS was determined by monitoring the conversion of L-[14C]arginine to L-[14C]citrulline. Reaction mixtures contained in a final volume of 50 µl, 50 mM Na+ Hepes buffer, pH 7.4, 100 µM EDTA, 0.2 mM CaCl2, 10 µg/ml calmodulin, 100 µM dithiothreitol, 50 µM BH4, 1.0 µM FAD, 1.0 µM FMN, 100 µg/ml bovine serum albumin, 500 µM NADPH, L-[14C]arginine as indicated, L-VNIO as indicated, and nNOS or eNOS. Reaction mixtures for iNOS were similar, but CaCl2 and calmodulin were omitted. Reaction was initiated by the addition of enzyme, and the solutions were maintained at 25 °C for 4 min. Reaction mixtures were quenched by the addition of 200 µl of stop buffer containing 100 mM Na+ Hepes buffer, pH 5.5, and 5 mM EGTA. Those samples were heated in a boiling water bath for 1 min, chilled and centrifuged. A portion (225 µl) of the supernatant was applied to small Dowex 50 columns (Na+ form, 1 ml resin), and the product L-[14C]citrulline was eluted with 2 ml of water and quantitated by liquid scintillation counting.
Optical Difference Spectroscopy-- Studies were carried out on a Shimadzu model 2501 dual beam UV-visible spectrophotometer using either nNOS as isolated (~20% low spin heme) or nNOS pretreated with imidazole (100% low spin heme). In the former case, 0.5 ml of nNOS as isolated (432 µg) in 50 mM Tris-HCl buffer, pH 7.5, 10% glycerol, and 0.1 mM EDTA was placed in the sample and reference cuvettes at 15 °C, and the base-line spectrum was adjusted to zero. Sequential samples of buffer and L-VNIO in buffer were added to the reference and sample cuvettes, respectively, and optical difference spectra at increasing concentrations of L-VNIO were obtained. Similar studies using imidazole in place of L-VNIO were carried out to determine the Ks for that ligand (KsImid = 86.2 µM, data not shown). The effect of L-VNIO on imidazole liganded nNOS was then determined by initially adding 1.0 mM imidazole to the cuvettes, setting the base line to zero, and then adding sequential samples of buffer and L-VNIO in buffer to the reference and sample cuvettes, respectively, as described above. For these studies KsL-VNIO was calculated from the relationship Ks(app)L-VNIO = KsL-VNIO (1 + [Imid]/KsImid), where KsL-VNIO is the true binding constant for L-VNIO, Ks(app)L-VNIO is the apparent binding constant for L-VNIO determined in the presence of imidazole, [Imid] is the concentration of imidazole (1.0 mM) and KsImid is the binding constant for imidazole as determined in the preliminary study mentioned (i.e. 86.2 µM).
Irreversible Inactivation of NOS--
Time- and
concentration-dependent inactivation kinetics for nNOS and
eNOS treated with various inhibitors were determined at 25 °C in
reaction mixtures (final volume = 0.15 ml) containing 50 mM Na+ Hepes buffer, pH 7.4, 0.1 mM
EDTA, 50 µM BH4, 2.0 mM
glutathione, 1.0 µM FAD, 1.0 µM FMN, 1 mg/ml bovine serum albumin, 100 units of superoxide dismutase, 0.2 mM CaCl2, 10 µg/ml calmodulin, 1.0 mM NADPH, L-arginine or inhibitor as indicated,
and ~40 µg NOS. Residual activity was determined after various time
intervals by adding a 25-µl aliquot of the reaction mixture to a
cuvette containing, in a final volume of 0.5 ml, 50 mM
Hepes buffer, pH 7.4, 0.1 mM EDTA, 50 µM
BH4, 10 µg/ml calmodulin, 0.2 mM
CaCl2, 0.1 mM GSH, 1.0 µM FAD,
1.0 µM FMN, 1 mg/ml bovine serum albumin, 0.5 mM NADPH, and 0.25 mM of L-arginine
and 5 µM bovine oxyhemoglobin (prepared by reduction with
sodium dithionite followed by gel filtration). Reaction mixtures for
iNOS were similar but lacked CaCl2 and calmodulin. Nitric
oxide-mediated oxidation of oxyhemoglobin was monitored at 401 nm ( = 0.038 µM
1) (47); the reference cuvette
contained a similar mixture without enzyme. The rate of NO formation
was determined and used to calculate the residual activity.
Determination of Heme Loss-- The ability of carbon monoxide (CO) to bind to the reduced heme cofactor of NOS and elicit a characteristic absorption maxima at 443 nm was used to determine the loss of heme cofactor after inactivation of nNOS by L-VNIO. The incubation conditions were similar to those used to determine irreversible inactivation except the final volume was 1.8 ml. After specific time intervals (0, 10, and 20 min), an aliquot (200 µl) of the inactivation reaction mixture was added to both sample and reference cuvettes containing 50 mM Na+ Hepes buffer, pH 7.4, and 10% glycerol to give final volume of 0.5 ml. The buffer in the sample cuvette was then saturated with CO, and the difference spectrum between 400 and 500 nm was determined (48).
Determination of NOS-mediated Cytochrome c
Reduction--
Reaction mixtures of 500 µl contained 50 mM Na+ Hepes buffer, pH 7.4, 100 µM EDTA, 50 µM NADPH, 50 µM
bovine heart cytochrome c, and nNOS.
NADPH-dependent reduction of cytochrome c was
monitored at 550 nm ( = 0.021 µM
1).
Where indicated 10 µM L-VNIO, 0.2 mM CaCl2, 10 µg/ml calmodulin, and/or 800 units/ml superoxide dismutase were added to the reaction mixtures.
Determination of NOS-mediated NADPH Oxidation--
The rate of
NADPH oxidation by NOS was determined spectrophotometrically by
monitoring the decrease in absorbance at 340 nm with time ( = 6.22 mM
1). The reaction mixtures contained in a
final volume of 0.5 ml 50 mM Na+ Hepes buffer,
pH 7.4, 0.1 mM EDTA, 50 µM BH4,
2.0 mM GSH, 1.0 µM FAD, 1.0 µM
FMN, 1 mg/ml bovine serum albumin, 0.2 mM
CaCl2, 10 µg/ml calmodulin, 0.25 mM NADPH,
where indicated 10 µM L-VNIO, and nNOS. NADPH
oxidation was initiated by addition of enzyme.
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RESULTS |
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Inhibition of NOS Isoforms by L-VNIO and Related
Compounds--
All NOS isoforms are inhibited by a variety of
L-arginine analogs that compete with L-arginine
for the amino acid binding site; we have reported previously that
N-alkyl-L-arginines with
n-alkyl substituents up to 4 carbons and the isosteric
N5-(1-iminoalkyl)-L-ornithines
are good to excellent inhibitors (24). Consistent with these findings,
in initial rate studies L-VNIO was found to be a potent
inhibitor of nNOS, and its binding was competitive with
L-arginine (Fig. 2). A replot
of the data shows that KiL-VNIO
is ~100 nM (Fig. 2, inset); this value is
substantially lower than the Km for
L-arginine (1.4 µM). Similar studies showed
L-VNIO also inhibits eNOS and iNOS competitively with
respect to L-arginine, but the Ki values
are much higher (i.e. 12.0 µM for eNOS and 60 µM for iNOS) (Table I).
Note that the KiL-VNIO/KmL-Arg
ratios for nNOS, eNOS, and iNOS are 0.07, 3.33 and 4.80, respectively, indicating that L-VNIO competes for the
L-arginine binding site of nNOS particularly well (Table
I). D-Arginine is neither a substrate nor an inhibitor of
NOS (1), and none of the NOS isoforms was inhibited by
D-VNIO when tested at 100 µM in the presence
of 20 µM L-arginine (data not shown).
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Effect of L-VNIO Binding on the Heme Spectrum of
nNOS--
A variety of studies indicate that the reactive guanidinium
nitrogen of substrate L-arginine is bound near the iron of
the NOS heme cofactor;
N-hydroxy-L-arginine is
then formed when that guanidinium nitrogen is oxidized by
O2, which has been bound and activated as a sixth, axial
heme iron ligand (1, 2). Although L-arginine does not bind
near enough to heme iron to act as a sixth axial ligand (49), other
inhibitors such as L-thiocitrulline do covalently interact
with heme iron (24, 50, 51). Such interactions can be revealed by
optical difference spectroscopy in which perturbations of the heme
spectrum caused by substrates or inhibitors are determined (49). As
shown in Fig. 3A, increasing
concentrations of L-VNIO, when added to solutions of native
nNOS, cause a type I difference spectrum. This result, which is similar
to that seen with L-arginine, indicates that
L-VNIO does not interact covalently with heme iron but does
bind sufficiently close to its sixth axial position to displace an
endogenous heme ligand (the identity of the ligand displaced is
presently unknown). The displacement of the endogenous ligand, which is
present in ~20% of nNOS as isolated (49), is responsible for the
spectral change shown in Fig. 3A. Fig. 3B is a
replot of the data in Fig. 3A showing that the
L-VNIO dissociation constant,
KsL-VNIO, is 1.1 µM; the previously reported value for
KsL-Arg is 2.5 µM
(49). We also determined KsL-VNIO
using imidazole-saturated nNOS. These studies confirmed that L-VNIO gives a type I optical difference spectrum and
provide a potentially more accurate estimate of
KsL-VNIO since the signal is
larger (i.e. heme iron is initially 100% low spin) and the
amounts of L-VNIO added are larger, making it unnecessary
to correct for L-VNIO bound to nNOS. With imidazole saturated nNOS, KsL-VNIO,
calculated as described under "Methods," was 1.4 µM
(data not shown). Note that
KsL-VNIO is a simple binding
constant measured in the absence of NADPH whereas
KiL-VNIO (Table I) is determined
under conditions of substrate turnover. The two values need not agree
(52).
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Inactivation of nNOS by L-VNIO--
The rate
measurements used to construct Fig. 2 were obtained immediately after
initiation of the enzymatic reaction. If the reaction mixtures were
monitored for several minutes, the progress curves were clearly concave
downward indicating occurrence of a progressive irreversible inhibition
of nNOS. Such inactivation was examined directly by incubating nNOS
with various concentrations of L-VNIO in the presence of
NADPH and monitoring the reaction mixtures for residual nNOS activity
at intervals (Fig. 4A). As shown, L-VNIO, but not D-VNIO, caused a
first-order inactivation of nNOS. There was no evidence of nNOS
reactivation in these studies; product formation was constant over the
time of the assay. In separate studies, passage of reaction mixtures
containing L-VNIO-inactivated nNOS through small
gel-filtration columns did not restore activity (not shown). A replot
of the data in Fig. 4A shows that the rate constant for
inactivation, kinact, is 0.078 min1 and KI, the equilibrium binding
constant of L-VNIO to nNOS prior to inactivation, is 90 nM (Fig. 4B). The latter value is similar to the
Ki measured under turnover conditions.
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Inactivation of nNOS by Related
N5-(1-Iminoalkyl)-L-ornithines and
N-Methyl-L-arginine--
Previous studies
established that nNOS as well as the other NOS isoforms are inhibited
and/or inactivated by L-NMA, the prototypic NOS inhibitor
(24, 28), and by L-NIO (24, 35, 36). In Fig.
5, inactivation of nNOS by these
compounds is compared with that seen with L-VNIO. As shown,
inactivation by 0.5 µM L-VNIO is comparable
to that seen with 2.0 µM L-NMA and greater
than that seen with 10 µM L-NIO. As was seen
with L-VNIO, inhibition by L-NIO is
NADPH-dependent; nNOS inactivation by L-NMA has
previously been shown to be NADPH-dependent (53, 54).
Interestingly, the next higher homolog of L-NIO,
methyl-L-NIO, is a much weaker inactivator of nNOS,
requiring a 10-fold higher concentration (100 µM) to
duplicate the inactivation seen with L-NIO. Further extension of the iminoalkyl group to form the saturated analog of
L-VNIO (i.e. ethyl-L-NIO) results in
a compound that does not inactivate nNOS under the conditions examined
(Fig. 5). Note that ethyl-L-NIO does bind competitively
with L-arginine and thereby inhibits nNOS (Table I); such
inhibition does not, however, progress to inactivation.
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Effect of L-VNIO on Other Reactions Catalyzed by nNOS-- In addition to NO synthesis, nNOS catalyzes the NADPH-dependent reduction of cytochrome c, an activity attributed to the reductase domain (1, 2). It also catalyzes the O2- and Ca2+/calmodulin-dependent, L-arginine-independent oxidation of NADPH, an activity requiring both the reductase and the oxygenase nNOS domains and resulting in the formation of superoxide (1, 2). As shown in Table III (experiments 1 and 2), L-VNIO has no effect on cytochrome c reduction but potently inhibits NADPH oxidation. That cytochrome c reduction is due to direct transfer of electrons to it from the reductase domain and not to formation of superoxide by the oxygenase domain is evident from experiments 3 and 4 showing that superoxide dismutase has no effect on the rate of cytochrome c reduction in the presence or absence of L-VNIO. In the absence of Ca2+/calmodulin the rate of electron flow within the reductase domain is greatly diminished and electrons do not flow from the reductase domain to the heme cofactor of the oxygenase domain (5). As shown in experiments 5-8, omission of Ca2+/calmodulin from the reaction mixtures decreases cytochrome c reduction ~ 96% and nearly eliminates NADPH oxidation. There is again no effect of L-VNIO and superoxide dismutase on cytochrome c reduction, confirming that this reductase domain specific reaction is not affected. The minimal residual NADPH oxidase activity seen in the absence of Ca2+/calmodulin (experiment 5) is apparently unrelated to that seen in the presence of Ca2+/calmodulin (experiment 1) since it is not inhibited by L-VNIO; it may reflect a very slow, normally undetectable formation of superoxide by the flavins of the reductase domain.
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Inactivation of nNOS by L-VNIO Results in Heme Loss-- Brief treatment of nNOS with dithionite or with NADPH in the presence of Ca2+/calmodulin reduces the heme cofactor from the Fe3+ to the Fe2+ state. With heme reduced, nNOS binds CO with a characteristic increase in absorption at 443 nm that is useful in quantitating bound, functional heme (1, 2). As shown in Table IV, treatment of nNOS with L-VNIO, but not D-VNIO, causes loss of the heme cofactor as judged by CO binding studies. Loss of heme closely parallels the loss of overall NOS activity indicating that loss of heme functionality or heme binding to the enzyme can account for virtually all mechanism-based inactivation.
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Ability of L-VNIO to Inactivate eNOS and iNOS-- As shown in Fig. 6, L-VNIO is a relatively weak inactivator of eNOS and it does not inactivate iNOS. Inactivation of eNOS is NADPH-dependent and follows first order kinetics, but even with a 20-fold higher concentration of L-VNIO the rate of inactivation of eNOS does not match that seen with nNOS (compare the rate of eNOS inactivation using 10 µM L-VNIO to that seen with nNOS using 0.5 µM L-VNIO). Detectable inactivation of iNOS did not occur even with 100 µM L-VNIO.
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DISCUSSION |
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Studies from this laboratory and others establish that all NOS
isoforms bind a variety of L-arginine and
L-homoarginine analogs. Initial binding is in all cases
competitive with L-arginine, and all of the analogs inhibit
all NOS isoforms if added in sufficiently high concentration (1, 24).
Three general mechanisms of inhibition and/or inactivation have been
distinguished. Some analogs such as L-canavanine (55),
N-cyclopropyl-L-arginine
(56), N
-nitro-L-arginine
with iNOS (57) and
N5-(1-iminobutyl)-L-ornithine
with nNOS (this work) bind to and dissociate from NOS rapidly but do
not react covalently; they are classic competitive inhibitors and do
not cause inactivation. L-Thiocitrulline is also in this
class, although it reversibly interacts to a limited degree (~20%)
as a covalent sixth axial ligand to the heme cofactor (50). "Slow
on-slow off" inhibitors comprise the second type of NOS inhibitors
and are exemplified by the slow binding and very slow dissociation of
N
-nitro-L-arginine from
nNOS and eNOS (58, 59). These inhibitors, which include the
S-alkyl-L-thiocitrullines (38, 39), are not
altered by the enzyme, but their binding is apparently accompanied by a
conformational change in NOS that is only slowly reversible.
Mechanism-based inhibitors (kcat inhibitors)
constitute the third type of NOS-inhibiting amino acids. Of these, the
best characterized is L-NMA, which has been shown to be
hydroxylated by nNOS and iNOS as a pseudosubstrate; that product,
N-hydroxy-N
-methyl-L-arginine,
undergoes further NOS-mediated reaction to form
CH3NO+, formaldehyde, NO, and citrulline by
complex mechanisms (53, 54). A presently unidentified intermediate in
this latter transformation apparently causes damage to and loss of the
NOS heme cofactor, but the product formed is not yet identified (60).
N
-Allyl-L-arginine (56)
and N
-amino-L-arginine
(34) have also been previously shown to cause mechanism-based,
irreversible inactivation of NOS, and there is a brief report that
L-NIO (35) (this work) causes similar inactivation. Inactivation by these compounds has not yet been mechanistically characterized. Although
N
-amino-L-arginine causes
somewhat more rapid inactivation of nNOS (kinact(max) = 0.35 min
1) (34),
neither it nor the other previously reported mechanism-based arginine
analog inactivators shows significant NOS isoform selectivity.
The present studies establish that L-VNIO is a potent, mechanism-based NOS inactivator with substantial nNOS selectivity. Thus, initial binding of L-VNIO to nNOS is followed by NADPH- and O2-dependent mechanism-based inactivation of the overall reaction and of the oxygenase domain-dependent NADPH oxidase reactions. The reductase domain-specific cytochrome c reduction activity in the presence or absence of Ca2+/calmodulin is not lost. Consistent with damage to the oxygenase domain, inactivation correlates with loss of the heme cofactor as judged by CO difference spectroscopy. Isoform selectivity derives both from differences in initial binding (i.e. the Ki values for eNOS and iNOS are 120- and 600-fold higher than that for nNOS) and from differences in subsequent enzyme-mediated activation of L-VNIO to a reactive derivative (i.e. iNOS is not subject to mechanism-based inactivation).
The precise mechanism by which L-VNIO is transformed by
nNOS (and presumably by eNOS, albeit more slowly) into an inactivating intermediate is not yet known. Previous studies establish that NOS-mediated metabolism of L-arginine and L-NMA
proceeds via cytochrome P450-like hydroxylation and oxidation reactions
(1, 2); in the case of L-NMA, carbon-centered free-radical
intermediates are formed (54). We designed L-VNIO with the
intent of developing an arginine analog that would place an allyl
moiety, known to be highly susceptible to free radical activation, in
close proximity to the NOS heme cofactor. The observations that initial
binding is competitive with L-arginine and that
steady-state Ki values are lower than or comparable
to the Km for L-arginine support the
view that L-VNIO binds to nNOS as an L-arginine
analog. The observation that a type I difference spectrum accompanies L-VNIO binding to imidazole liganded or native nNOS
indicates that a portion of the inhibitor binds closely enough to the
heme cofactor to displace (but not replace) imidazole or the endogenous sixth axial heme ligand present in nNOS as isolated. Because the binding site for the non-reactive guanidinium nitrogen of
L-arginine (i.e. the nitrogen not near the heme
iron and thus not hydroxylated or converted to NO) does not accomodate
groups larger than =NH or -NH2
(1),3 we know further that it
is the allyl moiety of L-VNIO that must be near heme.
Several specific mechanisms for nNOS-mediated L-VNIO activation thus seem plausible. Activated oxygen bound to heme may
epoxidize the allyl moiety, forming
N5-(1-imino-3,4-epoxybutyl)-L-ornithine;
this species could then react with protein or heme nucleophiles in a
manner that destroys heme functionality or binding. Alternatively, by a
process similar to that described for L-NMA (54), the allyl
moiety of L-VNIO may lose H· to form a reactive
allyl radical that damages or derivatizes the heme cofactor. Finally,
we have considered the possibility that -electrons of the allyl
moiety attack the highly electrophilic perferryl heme species that
normally hydroxylates L-arginine to form
N
-hydroxy-L-arginine (1,
2); this reaction would generate a highly reactive carbocation at the
terminus of L-VNIO. Although additional studies are
necessary to confirm which of these or related mechanisms is(are)
operative, we note that all of the proposed mechanisms depend
critically on the unsaturation of the L-VNIO side chain.
The mechanistic proposals are thus consistent with our observation that
the saturated analog, ethyl-L-NIO, does not inactivate
nNOS.
In summary, the present studies establish L-VNIO as the first neuronal isoform selective, mechanism-based amino acid inactivator of NOS. Our studies show further that selectivity can be achieved both in initial binding and in subsequent enzyme-mediated activation. The latter observation is of particular interest. Whereas all NOS isoforms catalyze the same overall reaction for L-arginine, our results indicate the isoforms can differ qualitatively as well as quantitatively in their ability to metabolize and thereby activate L-arginine analogs. There is thus the possibility of designing mechanism-based inhibitors that will have extremely high levels of isoform selectivity.
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ACKNOWLEDGEMENT |
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We thank Michael A. Hayward for excellent technical assistance and Christopher Frey for preparation of nNOS.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health grant DK48423.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
Medical College of Wisconsin, Milwaukee, WI 53226. Tel.: 414-456-8435;
Fax: 414-456-6510; E-mail: griffith{at}post.its.mcw.edu.
1
The abbreviations used are: NOS, nitric oxide
synthase; nNOS, neuronal (type I) NOS; eNOS, endothelial (type III)
NOS; iNOS, inducible (type II) NOS; NO, nitric oxide;
L-VNIO, vinyl-L-NIO (i.e.
N5-(1-imino-3-butenyl)-L-ornithine);
D-VNIO, vinyl-D-NIO (i.e.
N5-(1-imino-3-butenyl)-D-ornithine),
L-NMA,
N-methyl-L-arginine;
L-NIO, L-NIO,
N5-(1-iminoethyl)-L-ornithine;
BH4, (6R)-5,6,7,8-tetrahydrobiopterin; methyl-L-NIO,
N5-(1-iminopropyl)-L-ornithine;
ethyl-L-NIO,
N5-(1-iminobutyl)-L-ornithine;
FAB, fast atom bombardment; MS, mass spectrometry.
2 L. J. Roman and B. S. S. Masters, unpublished results.
3 B. R. Babu and O. W. Griffith, unpublished results.
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REFERENCES |
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