From the Institut für Pharmakologie und
Toxikologie, Karl-Franzens-Universität Graz,
Universitätsplatz 2, A-8010 Graz, Austria,
§ Department of Clinical Biochemistry, National Hospital for
Neurology and Neurosurgery, Queen Square, London WC 1N 3BG, United
Kingdom, and ¶ Institut für Medizinische Chemie und
Biochemie, Universität Innsbruck, Fritz-Pregl-Strasse 3, A-6020
Innsbruck, Austria
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tetrahydrobiopterin
((6R)-5,6,7,8-tetrahydro-L-biopterin
(H4biopterin)) is an essential cofactor of nitric-oxide
synthases (NOSs), but its role in enzyme function is not known. Binding of the pterin affects the electronic structure of the prosthetic heme
group in the oxygenase domain and results in a pronounced stabilization
of the active homodimeric structure of the protein. However, these
allosteric effects are also produced by the potent pterin antagonist of
NOS, 4-amino-H4biopterin, suggesting that the natural
cofactor has an additional, as yet unknown catalytic function. Here we
show that the 5-methyl analog of H4biopterin, which does
not react with O2, is a functionally active pterin cofactor
of neuronal NOS. Activation of the H4biopterin-free enzyme occurred in a biphasic manner with half-maximally effective
concentrations of approximately 0.2 µM and 10 mM 5-methyl-H4biopterin. Thus, the affinity of
the 5-methyl compound was 3 orders of magnitude lower than that of the
natural cofactor, allowing the direct demonstration of the functional
anticooperativity of the two pterin binding sites of dimeric NOS. In
contrast to H4biopterin, which inactivates nitric oxide
(NO) through nonenzymatic superoxide formation, up to 1 mM
of the 5-methyl derivative did not consume O2 and had no
effect on NO steady-state concentrations measured electrochemically with a Clark-type NO electrode. Therefore, reconstitution with 5-methyl-H4biopterin allowed, for the first time, the
detection of enzymatic NO formation in the absence of superoxide or NO
scavengers. These results unequivocally identify free NO as a NOS
product and indicate that reductive O2 activation by the
pterin cofactor is not essential to NO biosynthesis.
Nitric oxide is formed from the guanidino group of
L-arginine by nitric oxide synthases
(NOSs)1 (EC 1.14.13.39; Refs.
1 and 2). The neuronal (nNOS) and endothelial isoforms are
constitutively expressed and require micromolar free Ca2+
for activity, whereas the isoform first described in murine macrophages is cytokine-inducible and Ca2+-independent (inducible NO
synthase (type II)). The enzymes consist of an oxygenase domain with a
prosthetic heme group and a flavin-containing reductase domain
shuttling reducing equivalents from the cosubstrate NADPH to the heme.
Unlike other P450s, NOS requires H4biopterin as a cofactor,
but the actions of the pterin that underlie this requirement remain a
puzzle (3). It has been suggested to participate as a reactant in the
enzymatic hydroxylation of L-arginine to
NG-hydroxy-L-arginine and/or the
subsequent oxidation of the hydroxylated intermediate to NO and
L-citrulline (4-7). However, the allosteric effects of
H4biopterin binding are more obvious and have been more
accessible to experimentation. They involve changes in the conformation
of the protein around the heme and substrate binding pocket (8),
facilitate the binding of L-arginine (9), and are coupled
to a transition of the heme from a low spin, hexacoordinate state to a
high spin, pentacoordinate state (10-12). Also, the binding of
H4biopterin promotes dimerization of the enzyme. In inducible NO synthase (type II), it increases the level of dimerization per se (13), and in all NOS isozymes, it induces a form of
the dimer that is unusually resistant to dissociation by SDS (14). The
time course of conversion from low spin heme to high spin heme
correlates closely with enzyme activation (12); also, only dimeric
forms of the enzyme have been found to be active (15). Thus, it could
be supposed that the allosteric effects of H4biopterin fully explain the pterin dependence of NOS.
However, several pterin derivatives, such as dihydrobiopterin (9, 14,
16) or the 4-amino analog of H4biopterin (17, 18), produce
the same allosteric effects as the natural cofactor but do not support
NO synthesis. Inhibition of NOS by the oxidation-resistant deaza analog
of 6-methyltetrahydropterin (19), as well as by the potent
dihydropteridine reductase inhibitor 4-amino-H4biopterin (20), indicated that classical two-electron redox cycling of the pterin
cofactor may be essential for NO synthesis. This view was supported by
a recent report showing that although both the di- and tetrahydro forms
of various pterin derivatives have binding capacity, NOS activation was
only observed in the presence of the tetrahydro compounds (16).
Finally, a study of reaction intermediates indicated that
H4biopterin was necessary for reductive activation of the
oxy-ferrous complex of nNOS (21). Taken together, these data provide
circumstantial evidence for an essential role of the pterin redox
properties in NOS catalysis. In the crystal structure of dimeric
inducible NO synthase (type II) oxygenase, the pterin is located
proximal to the heme, without apparent access to the distal site where
O2 activation and substrate oxidation take place (8). Thus,
chemical contributions of the pterin to NOS catalysis are only likely
if they occur via the heme.
Another interesting feature of pterin-NOS interactions is
the highly anticooperative binding of H4biopterin to two
identical binding sites of homodimeric nNOS (12). The biphasic
association kinetics of [3H]H4biopterin
binding to a pterin-deficient form of the protein pointed to a
difference of at least 1,000-fold in the affinities for the binding of
the first and the second H4biopterin molecule, respectively. From the rapid association of
[3H]H4biopterin, the KD
value of the high affinity site was calculated to be less than 1 nM (12, 22). This high binding affinity probably explains
why isolated nNOS always contains about 1 equivalent of tightly bound
H4biopterin per dimer (23, 24).
The anticooperative binding of H4biopterin appears to have
important functional consequences because the pterin is essential for
the coupling of NADPH oxidation to L-arginine metabolism
(25). Thus, even in the presence of saturating L-arginine
concentrations, activation of pterin-deficient nNOS by
Ca2+/calmodulin results in the NADPH-dependent
reduction of O2 to O In the present study, we have reconstituted pterin-deficient nNOS with
the low affinity binding pterin derivative
5-methyl-H4biopterin. Unlike H4biopterin, this
compound proved not to react with O2. This property
allowed, for the first time, the detection of the enzymatic formation
of free NO in the absence of O Materials--
Purified rat nNOS containing approximately 1 equivalent of H4biopterin per dimer
(H4B(+)nNOS) was obtained from recombinant baculovirus-infected Sf9 cells as described previously (32). Pteridine-deficient nNOS containing <0.1 equivalent of
H4biopterin (H4B( Purity of 5-Methyl-H4biopterin--
The batch of
5-methyl-H4biopterin used in the present study did not
contain any detectable contaminating H4biopterin. The detection limit of the applied high performance liquid chromatography method with electrochemical detection (35) was 33 nM
H4biopterin in 1 mM solutions of
5-methyl-H4biopterin. Accordingly, the amount of
contaminating H4biopterin was Determination of NOS Activity--
NOS activity was determined
as the formation of L-[2,3,4,5-3H]citrulline
from L-[2,3,4,5-3H]arginine (36). Incubations
were for 10 min at 37 °C in 0.1 ml of 50 mM potassium
phosphate buffer, pH 7.4, containing 0.3 µg of nNOS, 0.1 mM L-[2,3,4,5-3H]arginine
(~60,000 counts/min), 0.2 mM NADPH, 10 µg/ml
calmodulin, 0.5 mM CaCl2, 0.2 mM
CHAPS, and pteridines as indicated. Of note, standard incubation
conditions were modified by omitting exogenous flavins that have been
recognized as potential unwanted sources of superoxide (37, 38).
H4B(+)nNOS and H4B(
The stoichiometry of NADPH oxidation and L-citrulline
formation was determined with H4B(+)nNOS under slightly
modified conditions, i.e. in the presence of 0.2 mM [3H]arginine, SOD (5,000 units/ml), and a
limiting concentration of NADPH (10 µM). The amount of
[3H]citrulline formed was measured after 3 h of
incubation at 37 °C.
Electrochemical Determination of NO and O2--
NO
and O2 were measured at 37 °C with commercially
available Clark-type electrodes (Iso-NO and ISO2; World
Precision Instruments, Berlin, Germany) (29). The electrodes were
connected to an Apple Macintosh computer via an analog to digital
converter (MacLab; AD Instruments Ltd., Hastings, United Kingdom), and
the output current was recorded under constant stirring. The NO
electrode was calibrated daily with acidified nitrite according to the
recommendations of the manufacturer. Two-point calibration of the
O2 electrode was performed with argon and air-saturated
distilled water, respectively. O2 consumption was measured
at 37 °C in sealed 1.8-ml vials completely filled with 50 mM potassium phosphate buffer, pH 7.4. The observed rates
(Kobs) were divided by the O2 and pterin
concentrations to obtain the respective second-order rate constants.
For the measurement of NO oxidation, a solution of authentic NO was
added to 0.5 ml of 50 mM potassium phosphate buffer, pH
7.4, in open glass vials at 37 °C. For the determination of
enzymatic NO formation, purified nNOS (5 µg) was incubated in 0.5-ml
open glass vials at 37 °C in 50 mM phosphate buffer, pH
7.4, containing 0.1 mM L-arginine, 0.2 mM NADPH, 10 µg/ml calmodulin, 0.5 mM
CaCl2, and variable concentrations of
H4biopterin or 5-methyl-H4biopterin. Pterins
were added as 100-fold concentrated aqueous solutions. Where indicated,
SOD was added to give a final concentration of 1,000 units/ml.
Experiments were terminated by the addition of hemoglobin (4 µM, final concentration). Specific enzyme activity was
calculated from the initial linear rise in the NO concentrations.
H4biopterin Binding--
Saturation binding
experiments using [3H] H4biopterin as a
high affinity radioligand were performed as described previously with
minor modifications (9). H4B( Reactivity of 5-Methyl-H4biopterin toward
Oxygen--
To test the reactivity of 5-methyl-H4biopterin
toward O2, we measured the nonenzymatic consumption of
O2 by H4biopterin and its 5-methyl analog. As
shown in Fig. 1A,
H4biopterin led to a consumption of O2 that was
linearly dependent on the concentration of the pterin (0.1-0.5
mM). Assuming a first-order rate law with respect to
O2, the calculated second-order rate constant for the reaction was 4.72 ± 0.44 M Activation of NOS by 5-Methyl-H4biopterin--
The
interesting chemical properties of 5-methyl-H4biopterin led
us to investigate its effect on NOS activity. As shown in Fig.
2, the presence of
5-methyl-H4biopterin resulted in a biphasic stimulation of
L-citrulline formation by H4B(
Determination of the stoichiometry of NADP+ and
L-citrulline formation by H4B(+)nNOS showed
that NADPH oxidation was largely uncoupled from L-arginine
oxidation in the absence of 5-methyl-H4biopterin or at low
concentrations (up to 1 µM) of
5-methyl-H4biopterin (NADP+/L-citrulline = 2.2 ± 0.2;
n = 4). The presence of 0.1 mM
5-methyl-H4biopterin led to an almost complete coupling of
the reactions, as indicated by a
NADP+/L-citrulline stoichiometry of 1.59 ± 0.07 (n = 4), which is close to the theoretical
value of 1.50 (5, 6).
Effect of 5-Methyl-H4biopterin on
[3H]H4biopterin Binding to
nNOS--
Radioligand binding experiments using
[3H]H4biopterin as a high affinity ligand (9)
showed that 5-methyl-H4biopterin bound specifically to the
pterin site of nNOS. As illustrated in Fig. 3, the 5-methyl analog displaced
[3H]H4biopterin from H4B( Effect of 5-Methyl-H4biopterin on NO Formation by
nNOS--
The inactivity of 5-methyl-H4biopterin toward
O2 suggested that incubation of nNOS with an excess of this
synthetic pterin should not lead to the inactivation of enzymatically
produced NO, as observed with the natural cofactor. Indeed, as shown in Fig. 4, significant NO formation was
detectable when H4B(
The concentration-dependent effects of
5-methyl-H4biopterin on NO formation by
H4B( Compared with the parent compound H4biopterin, the
5-methyl analog exhibited markedly reduced activity toward
O2, which may be due to steric hindrance by the 5-methyl
group, preventing the formation of the presumed hydroperoxide
intermediate at the C4a position of the pterin ring. Nevertheless,
5-methyl-H4biopterin stimulated pterin-deficient nNOS in a
biphasic manner, an effect that most likely reflects binding of the
pterin to the high and low affinity sites of the dimeric protein. The
KD for binding the natural cofactor,
H4biopterin, to the high affinity site is too low to be
determined in binding studies, but from the kinetics of
[3H]H4biopterin association to
H4B( Because the precise KD value for high affinity
H4biopterin binding is unknown, it was not possible to
convert the IC50 values for inhibition of
[3H]H4biopterin binding by the 5-methyl
analog into reliable KI values. However, based on
the solid evidence that the H4biopterin binding constant is
There are numerous reports demonstrating NO release from intact cells,
but it has proven difficult in the past to identify NO as a product of
the NOS reaction. Thus far, NO formation by isolated NOS has only been
detected in the presence of scavengers of either NO or superoxide.
Thus, enzymatic NO formation was apparent when NOS activity was assayed
as a hemoglobin-to-methemoglobin conversion (7) or in spin trapping
electron paramagnetic resonance spectroscopy experiments performed with
excess Fe2+-N-methyl-D-glucamine
dithiocarbamate as a NO trap (37). Besides NO scavengers, the presence
of the O A great deal of published circumstantial evidence supports our early
hypothesis that one step of the NOS reaction resembles the
hydroxylation of aromatic amino acids, an enzymatic reaction in which
H4biopterin functions as a donor of electrons required for
reductive O2 activation (5). Thus, stimulation of NOS
activity is only observed with fully reduced, i.e.
tetrahydro, pterins, whereas dihydro compounds are inhibitory (9, 16).
Furthermore, the redox-inactive N5-deaza analog of
6-methyl-tetrahydropterin was found to inhibit
H4biopterin-supported NOS activity, although the parent
compound was an active cofactor (19). Similarly, the 4-amino analog of
H4biopterin, an inhibitor of dihydropteridine reductase-catalyzed pterin redox cycling (20), potently inhibited both
steps of the NOS reaction, although it produced allosteric effects
identical to those of H4biopterin (17, 18). Finally, a
recent study demonstrated that the rate of the NOS reaction is
increased by binding of non-heme iron, indicating that an additional metal center might cooperate with H4biopterin in enzyme
catalysis (39). Nevertheless, compared with the finding that
5-methyl-H4biopterin does not stimulate phenylalanine
hydroxylase,3 the present
data unequivocally show that the same redox chemistry of the pterin is
not required in the NOS reaction. This constitutes conclusive
functional evidence that the pterin in NOS is not the site of
O2 activation. Although the redox potential of
5-methyl-H4biopterin is different from that of
H4biopterin,4 the
5-methyl compound can still undergo certain redox reactions that are
currently being investigated in our laboratory. Thus, at the present
time, we cannot rule out that the pterin cofactor, in addition to its
allosteric functions, interacts with the heme or another metal center
of NOS through an as yet unrecognized redox chemistry.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 and, eventually,
H2O2 (12). Because the two catalytic domains of
dimeric NOS operate independently (22, 26, 27), these results suggest
that native nNOS containing 1 equivalent of H4biopterin per
dimer generates NO and O
2 at the same time, two species known to react with each other very rapidly to yield peroxynitrite (28). Indeed, nNOS was found to produce free NO only in the presence of the
O
2 scavenger SOD (29). Inhibition of
calmodulin-dependent H2O2 formation
in the presence of relatively high, i.e. micromolar, concentrations of free H4biopterin suggests that enzymatic
O
2 formation is prevented by saturation of the low affinity
pterin binding site (12), but under these conditions, autooxidation of
free H4biopterin appears to produce sufficient O
2
to completely inactivate the NO formed by the pterin-saturated enzyme,
again rendering SOD essential to detect free NO (29). Because SOD was
reported to catalyze the interconversion of NO and nitroxyl anion
(NO
) under certain conditions (30), it was proposed that
the initial product of the NOS reaction is, in fact, nitroxyl that is
converted to free NO by SOD (31). Although we have not observed any
significant oxidation of nitroxyl released from Angeli's salt by SOD
under our experimental
conditions,2 the definitive
rebuttal of the nitroxyl hypothesis would require a demonstration of NO
formation by nNOS in the absence of SOD or other scavengers, a goal
that has not been achieved thus far.
2 or NO scavengers. In addition,
the previously calculated 3 orders of magnitude difference in the
affinity of the two highly anticooperative pterin binding sites becomes
visible in NOS activity assays with
5-methyl-H4biopterin.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)nNOS) was obtained from
Sf9 cells pretreated with 2,4-diamino-6-hydroxypyrimidine as
described previously (24). NO solutions were prepared as described
previously (33). L-[2,3,4,5-3H]Arginine
hydrochloride (57 Ci/mmol) was from American Radiolabeled Chemicals
Inc., purchased through Humos Diagnostica GmbH (Maria Enzersdorf,
Austria).
3'[3H](6R)-5,6,7,8-Tetrahydro-L-biopterin
(14 Ci/mmol) was prepared from [8,5'-3H]GTP as described
previously (34). Unlabeled pterins including 5-methyl-H4biopterin were from Dr. B. Schircks Laboratories
(Jona, Switzerland). Other chemicals including bovine Cu- and Zn-SOD were from Sigma.
0.0033%. Most of the
experiments shown here have been repeated with a batch of
5-methyl-H4biopterin containing approximately 0.2 µM H4biopterin in 1 mM solutions with identical results.
)nNOS exhibited a similar
specific activity of approximately 0.4 µmol
L-citrulline × mg
1 × min
1 under those conditions.
)nNOS and
H4B(+)nNOS (0.5 µg each) were incubated for 10 min at
37 °C with 10 nM
[3H]H4biopterin (~14 nCi) and increasing
concentrations of 5-methyl-H4biopterin (10
8
to 10
2 M) in 0.1 ml of a 50 mM
potassium phosphate buffer, pH 7.4, followed by vacuum filtration of
the protein using the MultiScreen Assay System (Millipore) and liquid
scintillation counting of bound radioactivity. Data were corrected for
nonspecific binding in the presence of 1 mM unlabeled
H4biopterin. IC50 values obtained from
individual experiments were used to calculate KI values according to the following equation: KI = IC50/(1 + [H4B]f/KD) with
[H4B]f corresponding to the concentration of free
H4biopterin (10 nM), and KD corresponding to the binding constant for H4biopterin (
1
nM) (12, 22).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
s
1 (n = 8) at 37 °C. In contrast to
H4biopterin, the 5-methyl analog did not trigger a
detectable consumption of O2 at concentrations of up to 1 mM. Because the reduction of O2 by
H4biopterin results in O
2-mediated oxidation of
NO (29), we tested the two pterins for their effects on the rate of NO
oxidation measured in O2-containing solutions using a
Clark-type NO electrode. Fig. 1B shows that H4biopterin (0.1 mM), but not its 5-methyl
analog, led to a rapid, apparently zero-order disappearance of the NO
signal. The effect of H4biopterin was completely inhibited
by SOD (1,000 units/ml; data not shown). The initial rate of NO
consumption by added H4biopterin (0.1 mM, final
concentration) was 38.5 ± 2.7 nM s
1
(n = 4) at 37 °C. This rate agrees well with the
O2 consumption data showing that the initial rate of
O
2 generation by 0.1 mM H4biopterin
was about 80 nM s
1 O
2 under
identical conditions.
View larger version (16K):
[in a new window]
Fig. 1.
Oxygen consumption (A) and
NO oxidation (B) by H4biopterin
and its 5-methyl analog. The consumption of O2
(A) and the oxidation of authentic NO (B) were
measured electrochemically with O2 and NO electrodes,
respectively, at 37 °C as described under "Experimental
Procedures." The final pterin concentrations added at the time points
shown are indicated by arrows. Results are original traces
representative of three experiments.
)nNOS. The
first phase occurred with an EC50 of approximately 0.2 µM, resulting in an increase of the specific activity
from a residual value of about 20 nmol L-citrulline × min
1 × mg
1 to 100 nmol × min
1 × mg
1 in the presence of 10 µM 5-methyl-H4biopterin. The enzyme activity approached the basal activity of H4B(+)nNOS (~150
nmol × min
1 × mg
1) and was not
further increased by up to 0.3 mM
5-methyl-H4biopterin. The basal activity of
H4B(+)nNOS was not significantly affected by up to 1 mM 5-methyl-H4biopterin. The second phase of
enzyme activation occurred with a calculated EC50 of about
10 mM 5-methyl-H4biopterin with both
H4B(+)nNOS and H4B(
)nNOS.
View larger version (21K):
[in a new window]
Fig. 2.
Effect of
5-methyl-H4biopterin on nNOS-catalyzed
L-citrulline formation. Purified
H4B(+)nNOS ( ) and H4B(
)nNOS (
) (0.2 µg each) were incubated for 10 min at 37 °C in 0.1 ml of 50 mM triethanolamine/HCl buffer, pH 7.4, containing 0.1 mM L-[2,3,4,5-3H]arginine
(~80,000 counts/min), 0.2 mM NADPH, 0.5 mM
CaCl2, 10 µg/ml calmodulin, 2.4 mM
2-mercaptoethanol, and 0.2 mM CHAPS in the presence of the
indicated concentrations of 5-methyl-H4biopterin. Data are
the mean values ± S.E. of three experiments.
)nNOS
and H4B(+)nNOS with IC50 values of 78 ± 7.1 and 62 ± 1.3 µM (means ± S.E.;
n = 3), respectively. Because the KD
of the low affinity site is too high to be observed in radioligand
binding experiments, 5-methyl-H4biopterin most likely
displaced H4biopterin bound with high affinity to H4B(+)nNOS. Based on the KD of
1
nM for high affinity binding of H4biopterin
(12, 22), the maximal KI values for
5-methyl-H4biopterin binding to H4B(
)nNOS and
H4B(+)nNOS were calculated to be 7.2 and 5.5 µM, respectively.
View larger version (21K):
[in a new window]
Fig. 3.
Effect of
5-methyl-H4biopterin on
[3H]H4biopterin
binding. Purified H4B(+)nNOS ( ) and
H4B(
)nNOS (
) (0.5 µg each) were incubated for 30 min
at 25 °C with 10 nM
[3H]H4biopterin (~14 nCi) in 0.1 ml of a 50 mM triethanolamine/HCl buffer, pH 7.4, in the presence of
increasing concentrations of 5-methyl-H4biopterin
(10
8 to 10
2 M) followed by
liquid scintillation counting of protein-bound radioactivity. Data
(means ± S.E.; n = 3) were corrected for
nonspecific binding in the presence of 1 mM unlabeled
H4biopterin.
)nNOS was incubated with
5-methyl-H4biopterin (1 mM; Fig. 4A)
instead of the natural cofactor (0.1 mM; Fig.
4B). The specific enzyme activity calculated from the
initial linear increase in the NO concentration was 52 ± 3 nmol × mg
1 × min
1, giving a
steady-state concentration of 0.20 ± 0.05 µM NO
(means ± S.E.; n = 3). The addition of SOD (1,000 units/ml) further increased this level to 0.31 ± 0.09 µM NO. The response of the electrode was completely
abolished by the addition of the NO scavenger hemoglobin (4 µM, final concentration). As shown in Fig. 4B,
the enzyme did not produce any detectable NO in the presence of 0.1 mM H4biopterin without SOD. The addition of SOD
(1,000 units/ml) led to the formation of NO with an apparent specific
activity of 45 ± 12 nmol NO × mg
1 × min
1 and a steady-state NO concentration of 0.29 ± 0.05 µM (means ± S.E.; n = 3).
Again, the signal returned to baseline after the addition of hemoglobin
(4 µM, final concentration). Similar results were
obtained with H4B(+)nNOS (data not shown).
View larger version (20K):
[in a new window]
Fig. 4.
Effects of
5-methyl-H4biopterin and SOD on the formation of
NO by nNOS. Purified H4B( )nNOS (10 µg) was
incubated at 37 °C in the presence of 1 mM
5-methyl-H4biopterin (A) or 0.1 mM
H4biopterin (B). NOS, SOD, and hemoglobin
(Hb) were added at the time points indicated by
arrows to give the indicated final concentrations. NO was
measured electrochemically with a Clark-type electrode as described
under "Experimental Procedures." Results are original traces
representative of six similar experiments.
)nNOS and H4B(+)nNOS are summarized in Fig. 5. With both preparations,
half-maximal enzyme activation was observed at 5-10 µM
5-methyl-H4biopterin. The maximal specific activities of
H4B(
)nNOS and H4B(+)nNOS calculated from the
initial rates of NO formation at 1 mM of
5-methyl-H4biopterin were 52 ± 3 and 88 ± 4 nmol NO mg
1 × min
1 (means ± S.E.;
n = 3) respectively, values that are in good agreement with the rate of NO formation obtained with H4biopterin in
the same assay.
View larger version (22K):
[in a new window]
Fig. 5.
Concentration-dependent effect of
5-methyl-H4biopterin on NO formation by
nNOS. Purified H4B(+)nNOS ( ) and
H4B(
)nNOS (
) (10 µg each) were incubated in the
presence of the indicated concentrations of
5-methyl-H4biopterin. NO was measured electrochemically
with a Clark-type electrode as described under "Experimental
Procedures." Data are the mean values ± S.E. of three
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)nNOS, we have calculated that the binding constant
is
1 nM (12, 22). Because this value is about 1 order of
magnitude lower than the typical NOS assay concentrations (10-30
nM), biphasic pterin binding is not detectable in
functional studies using H4biopterin as a cofactor.
However, the 3 orders of magnitude lower affinity of the 5-methyl
derivative apparently allowed, for the first time, monitoring of the
saturation of the high affinity site in the arginine-to-citrulline
conversion assay. The 50,000-fold difference in the affinities of the
two binding sites (EC50 values of approximately 0.2 µM and 10 mM) agrees well with our earlier
estimate that the KD values for high and low
affinity binding of H4biopterin to dimeric nNOS differ by
at least 3 orders of magnitude (12, 22). Because the second phase of
enzyme activation required relatively high concentrations of
5-methyl-H4biopterin, it was conceivable that this effect
was mediated by contaminating H4biopterin. However, this is
unlikely because (i) the batch of 5-methyl-H4biopterin used
throughout this study did not contain any detectable
H4biopterin (
0.0033%), and (ii) virtually identical data
were obtained with a batch containing an at least 10-fold higher amount
of H4biopterin (0.03%).
1 nM (12, 22), we have calculated that the upper limit of
the KI value for the binding of
5-methyl-H4biopterin is approximately 5 µM.
This calculated binding constant is about 20-fold higher than the
EC50 value obtained in the arginine-to-citrulline conversion assay, indicating that the KD for high
affinity H4biopterin binding to nNOS may be as low as
50 pM.
2 scavenger SOD was also shown to switch the outcome
of the NOS reaction toward detectable free NO (29, 31). Even though
those results strongly favor the view that NOS does indeed form NO, it
has remained unclear whether or not NOS would also make NO in the
absence of scavengers which, with the exception of SOD, are not present
in cells. Our results demonstrate for the first time that nNOS forms
free NO in the absence of scavengers when the enzyme is reconstituted with the autooxidation-resistant 5-methyl analog of
H4biopterin instead of the natural cofactor. The results
unequivocally identify free NO as the initial product of the NOS
reaction and provide further evidence for our proposal that the failure
to detect NO formation by purified nNOS is a consequence of NO
inactivation by O
2 formed during autooxidation of exogenously
added H4biopterin (29). Unlike L-citrulline
formation, NO production was not stimulated in a biphasic manner but
gradually increased when the concentration of
5-methyl-H4biopterin was increased from 5 µM
to 1 mM. This concentration range is clearly between the
concentrations required for high affinity and low affinity activation
of L-citrulline formation (see Fig. 2). Interestingly, the
stoichiometry of NADP+ and L-citrulline
formation showed that the presence of 0.1 mM 5-methyl-H4biopterin was sufficient for virtually complete
coupling of NADPH and L-arginine oxidation. These results
may indicate that saturation of one pterin site of dimeric nNOS with
5-methyl-H4biopterin leads to inhibition of heme reduction
and associated superoxide formation by the pterin-free subunit.
Although this hypothesis appears to provide a reliable explanation for
the formation of free NO by the enzyme subsaturated with
5-methyl-H4biopterin, additional studies are needed to
settle the complex relationship between biphasic
5-methyl-H4biopterin binding and NOS activity.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants 11478 and 13013 (to B. M.), Grant 12191 (to K. S.), and Grant 11301 (to E. R. W.) of the Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich.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. Tel.:
43-316-380-5567; Fax: 43-316-380-9890; E-mail:
mayer{at}kfunigraz.ac.at.
2 K. Schmidt and B. Mayer, unpublished observation.
3 E. R. Werner, H.-J. Habisch, A. C. F. Gorren, L. Canevari, K. Schmidt, G. Werner-Felmayer, and B. Mayer, manuscript in preparation.
4 A. C. F. Gorren, A. J. Kungl, K. Schmidt, E. R. Werner, and B. Mayer, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NOS, nitric-oxide
synthase;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
H4biopterin, (6R)-5,6,7,8-tetrahydro-L-biopterin;
5-methyl-H4biopterin, N5-methyl-(6R)-5,6,7,8-tetrahydro-L-biopterin;
NO, nitric oxide;
nNOS, neuronal nitric-oxide synthase (type I);
H4B(+)nNOS, neuronal nitric-oxide synthase containing one
molecule of bound tetrahydrobiopterin per dimer;
H4B()nNOS, neuronal nitric-oxide synthase containing
0.1 equivalent of tetrahydrobiopterin;
SOD, superoxide
dismutase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|