From the Division of Hepatology and Gene Therapy, Department of Medicine, University of Navarra, 31008 Pamplona, Spain
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ABSTRACT |
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S-Adenosylmethionine serves as the
methyl donor for many biological methylation reactions and provides the
propylamine group for the synthesis of polyamines.
S-Adenosylmethionine is synthesized from methionine and ATP
by the enzyme methionine adenosyltransferase. The cellular factors
regulating S-adenosylmethionine synthesis have not been
well defined. Here we show that in rat hepatocytes S-nitrosoglutathione monoethyl ester, a cell-permeable
nitric oxide donor, markedly reduces cellular
S-adenosylmethionine content via inactivation of methionine
adenosyltransferase by S-nitrosylation. Removal of the
nitric oxide donor from the incubation medium leads to the
denitrosylation and reactivation of methionine adenosyltransferase and
to the rapid recovery of cellular S-adenosylmethionine
levels. Nitric oxide inactivates methionine adenosyltransferase via
S-nitrosylation of cysteine 121. Replacement of the acidic
(aspartate 355) or basic (arginine 357 and arginine 363) amino acids
located in the vicinity of cysteine 121 by serine leads to a marked
reduction in the ability of nitric oxide to S-nitrosylate
and inactivate hepatic methionine adenosyltransferase. These results
indicate that protein S-nitrosylation is regulated by the
basic and acidic amino acids surrounding the target cysteine.
In the liver S-adenosylmethionine
(AdoMet)1 serves as the
methyl donor for many biological methylation reactions (such as DNA, proteins, phospholipids, and adrenergic, dopaminergic, and
serotoninergic molecules) and provides the propylamine group for the
synthesis of polyamines (1-3). AdoMet is synthesized from methionine
and ATP by the enzyme methionine adenosyltransferase (MAT). There are
two MAT genes; one is expressed only in the liver, and the other is
expressed in extrahepatic tissues and fetal liver (2-4). Up to 85% of
all methylation reactions and as much as 50% of methionine metabolism
occur in the liver (5), which agrees with this tissue having the
highest specific activity of MAT (6). Moreover, in the liver the
half-life of AdoMet is of only about 5 min (6).
Reduced levels of AdoMet and/or MAT activity, resulting in the abnormal
metabolism of methionine, have been found in human cirrhosis and in a
variety of experimental models including liver injury induced by
ethanol, CCl4, and galactosamine (3, 7). The importance of
this alteration in AdoMet synthesis in the pathogenesis of a variety of
liver disorders is suggested by the finding that exogenous AdoMet
administration protects from the hepatotoxic effect induced by a
variety of agents, such as ethanol, CCl4, paracetamol,
tumor necrosis factor, and galactosamine (3, 7). The cellular factors
regulating hepatic AdoMet levels are beginning to be defined. One such
factor is nitric oxide (NO). In previous studies we demonstrated that
conditions that induce NO synthesis, such as septic shock and hypoxia,
induce the inactivation of hepatic MAT without affecting the expression
of the liver-specific MAT gene (8, 9). Further, we have reported
previously that incubation of rat hepatocytes with
S-nitrosoglutathione monoethyl ester (EGSNO), a
cell-permeable NO donor, induces MAT inactivation (10). We have also
shown that purified rat liver MAT is inactivated by incubation with NO
donors (3-morpholinosydnonimine,
S-nitroso-N-acetylpenicillamine, and
S-nitrosoglutathione) (8, 10). In addition, we have recently demonstrated that liver MAT is S-nitrosylated both in
vitro and in vivo (10), and further, we have identified
cysteine 121 as the site of molecular interaction of NO and liver MAT
(8). Because these results indicate that NO (or related molecules) is a
critical regulator of liver MAT activity, we were interested in whether
AdoMet content in hepatocytes is regulated by NO.
There are few studies trying to identify the active site features that
control protein S-nitrosylation. It has been recently proposed (11) that protein S-nitrosylation involves an
acid-base-catalyzed nitrosothiol (SNO)/SH exchange reaction, where the
target cysteine residue is localized next to basic and acidic amino
acids. Here, we were interested in determining the structural factors
that govern liver MAT S-nitrosylation and inactivation using
liver recombinant enzyme and mutants of MAT where the acidic and basic amino acids in the vicinity of cysteine 121 were replaced by serine by
site-directed mutagenesis. Recognition of the topology involved in
protein S-nitrosylation is likely to prove useful in
identifying new targets of protein S-nitrosylation.
Isolation and Incubation of Hepatocytes--
Hepatocytes were
isolated from normally fed Wistar rats (250 g) as described previously
(10). Isolated hepatocytes were incubated in the absence or presence of
EGSNO at 37 °C. At the indicated periods of time, 2 ml of the cell
suspension (2 × 106 cells/ml) were poured into
precooled centrifuge tubes and washed twice with phosphate/saline
buffer. Hepatocytes were then used for AdoMet or MAT activity
determinations. Cell viability was determined before and after
incubations by the trypan blue exclusion test. Only preparations with
viability over 85% were used.
AdoMet Measurements--
AdoMet concentration was determined by
high pressure liquid chromatography following the procedure described
by Fell et al. (12) modified by Miller et al.
(13). Samples of 2 × 106 hepatocytes washed twice in
phosphate/saline buffer were homogenized in 200 µl of 0.4 M perchloric acid. Homogenates were centrifuged at
10,000 × g and 4 °C for 15 min. 100 µl of the
supernatant were analyzed on a Bio-Sil® ODS-5S column
equilibrated in 0.01 M ammonium formate, 4 mM
heptanesulfonic acid, pH 4.0. Elution was carried out with a 31-min
linear gradient of acetonitrile (0-25%) in the same buffer.
Chromatograms were analyzed using the Beckman System Gold software.
Site-directed Mutagenesis; Purification and Characterization of
MAT Mutants--
Mutants are identified by a number that indicates the
residue in the sequence of the enzyme that has been replaced. MAT
mutants C121S, D355S, D355E, R357S, R363S, R363K, G359S, and
R357S/R363S were obtained by inverse polymerase chain reaction
according to the procedure of Serrano et al. (14) using the
plasmid pSSRL, which includes a 1.2-kilobase pair fragment containing
the rat liver MAT coding region (15). The mutants were identified by sequencing of the complete MAT cDNA. Expression and purification of
WT (wild type) and mutant proteins were carried out as described previously (15). Protein purity was greater than 95% in all preparations as determined by SDS-polyacrylamide gel electrophoresis. Protein concentration was measured using the Bio-Rad protein assay kit
based on the Bradford assay (16). The recombinant WT and MAT mutants
analyzed presented similar values of specific activity: 4.36 ± 0.28, 4.19 ± 0.72, 3.97 ± 0.91, 4.22 ± 0.31,
3.89 ± 0.74, 4.23 ± 0.41, 4.89 ± 0.52, 4.01 ±
0.67, and 4.12 ± 0.37 nmol/min/mg protein for WT, C121S, D355S,
D355E, R357S, R363S, R363K, G359S, and R357S/R363S, respectively. To
exclude that mutant proteins presented any major alteration in their
tertiary structure, fluorescence analysis was performed using a
Perkin-Elmer LS-5B fluorimeter. Emission spectra were recorded between
290 and 440 nm in 5 mM Tris, pH 7.4, with excitation
wavelength set at 280 nm. Protein concentration was 0.02 mg/ml. No
differences were observed when the spectra of recombinant WT and MAT
mutants were compared.
Determination of MAT S-Nitrosylation--
MAT
S-nitrosylation was measured according to the procedure
described by Ruiz et al. (10). Briefly, samples of purified recombinant MAT (0.4 mg/ml) were incubated with
S-nitrosoglutathione (GSNO) at 37 °C for 10 min. The
excess GSNO was removed by DEAE chromatography.
S-Nitrosylated proteins were incubated with 2.2 mM HgCl2, and the amount of NO released was
measured by the chemiluminescence derived from its reaction with ozone
using a Sievers NOA 280 nitric oxide analyzer. Standard curves were
obtained with known concentrations of GSNO.
MAT Activity Measurements--
MAT activity was measured as
described by Cabrero et al. (17) using saturating
concentrations of the substrates, 5 mM methionine and 5 mM ATP, in the absence of any reducing agent. Samples of 2 × 106 hepatocytes, prepared as mentioned above,
were washed twice in phosphate/saline buffer and homogenized by
freezing and thawing in 10 mM Tris/HCl, 0.3 M
sucrose, and 1 mM EGTA, pH 7.5, containing 0.1%
phenylmethylsulfonyl fluoride and 0.1% benzamidine. Cytosol was
obtained by centrifugation at 100,000 × g at 4 °C.
Excess EGSNO was removed by DEAE chromatography on a 1-ml column (10), and the enzyme activity was measured. Samples of purified WT and mutant
recombinant rat liver MAT were incubated for 10 min at 37 °C in the
presence or absence of GSNO or peroxynitrite. After incubation, excess
GSNO or peroxynitrite was removed by DEAE chromatography (10), and the
enzyme activity was measured.
Preparation of GSNO, EGSNO, and Peroxynitrite--
GSNO and
EGSNO were prepared according to Ruiz et al. (10). Briefly,
equimolar concentrations of an aqueous solution of NaNO2
and a freshly prepared GSH or EGSH solution in 250 mM HCl and 0.1 M EDTA, pH 1.5, were mixed. The resulting mixture
was incubated at room temperature for 5 min and then neutralized with NaOH. Peroxynitrite synthesis was carried out by the method described by Beckman et al. (18) modified by Pannala et al.
(19). Only freshly prepared solutions of GSNO, EGSNO, and peroxynitrite
were used.
Cytosolic Nitrite and Nitrate Measurements--
Cytosolic
nitrite and nitrate (NOx) were reduced to NO by incubation with 1 N HCl
containing 50 mM VCl3 (10). The resulting NO
was measured by the chemiluminescence derived from its reaction with
ozone using a Sievers NOA 280 nitric oxide analyzer (10).
NO Reduces AdoMet Content in Hepatocytes--
The effect of NO on
the content of AdoMet was examined in isolated rat hepatocytes.
Incubation of rat hepatocytes with the cell-permeable NO donor EGSNO (5 mM) induced a time-dependent reduction of the
cellular AdoMet content (Fig. 1). Within
15 min of the addition of EGSNO to the incubation medium, the
hepatocyte content of AdoMet decreased about 80%, i.e. from
1.03 ± 0.08 nmol of AdoMet/106 cells in control
hepatocytes to 0.22 ± 0.035 nmol of AdoMet/106 cells
in hepatocytes treated with the NO donor (Fig. 1). The reversibility of
the depletion of AdoMet by EGSNO in isolated rat hepatocytes was
examined by removing the NO donor from the incubation medium and
resuspending the cells in fresh buffer without EGSNO (Fig. 1). Within
15 min of the removal of the NO donor and resuspension of the cells in
fresh medium without EGSNO, cellular AdoMet content returned close to
that of untreated hepatocytes (Fig. 1). When hepatocytes were
maintained in the presence of the NO donor, the depletion of the
cellular content of AdoMet was maintained during the next 15 min (Fig.
1). As previously demonstrated (10), incubation with EGSNO within 15 min induced the inactivation of MAT (from 36.4 ± 0.5 pmol/min/mg
of protein in control hepatocytes to 10.95 ± 2.8 pmol/min/mg of
protein in the presence of 5 mM EGSNO). Removal of the NO
donor from the incubation medium returned MAT activity to values
similar to those of the untreated hepatocytes (29.18 ± 1.1 pmol/min/mg of protein) within 15 min.
We next analyzed whether the effect of EGSNO on AdoMet content in
hepatocytes was dose-dependent (Fig.
2). The addition of EGSNO (0.5-5
mM) to isolated rat hepatocytes induced a
dose-dependent accumulation of NOx, which led to a
progressive depletion of AdoMet content (Fig. 2). Significant reduction
of the AdoMet content in hepatocytes was already observed at 1 mM EGSNO, a condition where intracellular NOx increased
about 5-fold. The addition of EGSNO to isolated rat hepatocytes
induced, as described previously (10), a dose-dependent
inactivation of MAT activity (data not shown).
The Structural Factors That Govern Liver MAT
S-Nitrosylation--
S-Nitrosylation of recombinant WT rat
liver enzyme and mutants of MAT was measured after reaction with
various concentrations of GSNO (5-100 µM). In WT rat
liver, MAT incubation for 10 min with 100 µM GSNO
produced the S-nitrosylation of about 1.3 thiol residues/enzyme subunit (Fig. 3).
Incubation with GSNO also induced the inactivation of the enzyme. As
shown in Fig. 4, there is a close inverse
correlation between the extent of S-nitrosylation and loss
of activity induced by GSNO (r = 0.95, p < 0.001). Incorporation of 1 mol of SNO/mol of MAT
subunit led to about 80% inactivation of MAT. Maximal
S-nitrosylation and inactivation were obtained within 10 min
of incubation with GSNO (data not shown). We have previously shown (8)
that replacement of cysteine 121 by serine (referred to as C121S)
prevented the ability of NO donors to inactivate hepatic MAT.
Consistently, incubation of C121S with GSNO (5-100 µM)
resulted in the incorporation of only about 0.2 mol of (SNO) per mol of
MAT subunit. The mechanism of protein S-nitrosylation has
been recently proposed (11) to involve an acid-base-catalyzed SNO/SH
exchange reaction, where the target cysteine residue is located next to
basic and acid amino acids. In human and rat liver MAT, cysteine 121 is
not flanked by acid and basic amino acids but by glutamine and valine
(20-22). However, when the three-dimensional structure of liver MAT is
analyzed, one residue of aspartic acid (Asp-355) and two residues of
arginine (Arg-357, Arg-363) are all found in the vicinity of the sulfur
group of cysteine 121 (Fig. 5).
Replacement of aspartic acid 355 by serine (D355S) markedly reduced the
S-nitrosylation of the mutant enzyme in the presence of GSNO
(Fig. 3). Replacement of arginine 357 (R357S) or of arginine 363 (R363S) by serine led also to a marked reduction of the
S-nitrosylation of the mutant enzyme as compared with the WT
liver MAT (Fig. 3). Further, a double mutant, where arginine 357 and
arginine 363 were both replaced by serine (R357S/R363S) incorporated
only about 0.4 mol of SNO/mol of MAT subunit after incubation with 100 µM GSNO (Fig. 3). As negative controls, we have replaced
arginine 363 by lysine (R363K) and aspartic acid 355 by glutamic acid
(D355E) and observed that these conservative changes had no effect on the S-nitrosylation of the mutant enzymes by GSNO as
compared with the WT liver MAT. Fig. 3 shows the results obtained with the mutant enzyme R363K; similar results were observed with the mutant
enzyme D355E (data not shown). As an additional control we have changed
glycine 359, a non-charged residue that is separated by 5.3 Å from the
thiol group of cysteine 121, to serine (G359S) and observed that this
mutation had no effect on the S-nitrosylation of the mutant
enzyme by GSNO (data not shown). These controls provide evidence that
the effects on MAT S-nitrosylation of changing arginine 357, arginine 363, or aspartic acid 355 to serine are due to loss of charge
and not to alterations in the position of cysteine 121. Finally,
similarly to the WT recombinant MAT, incubation of mutants D355S,
D355E, R357S, R363S, R363K, G359S, and R357S/R363S with millimolar
concentrations of GSNO resulted in the incorporation of about 10 mol of
SNO/mol of MAT subunit, indicating that all cysteine residues present
in the protein (21) were S-nitrosylated.
We next analyzed whether these mutants showing impaired MAT
S-nitrosylation were resistant to GSNO-induced enzyme
inactivation. Whereas incubation of the WT recombinant enzyme with 50 µM GSNO led to 70% reduction of the enzyme activity,
replacement of cysteine 121 by a serine residue prevented the ability
of GSNO to inactivate liver MAT (Fig. 6).
Replacement of aspartic acid 355, arginine 357, or arginine 363 by
serine led to a marked reduction of the ability of GSNO to inactivate
liver MAT (Fig. 6). Further, replacement of both arginine 357 and
arginine 363 by serine was as effective as the substitution of
cysteine 121 by serine to prevent GSNO-induced MAT inactivation (Fig.
6). On the contrary, replacement of arginine 363 by lysine, aspartic
acid 355 by glutamic acid, or glycine 359 by serine did not affect the
ability of GSNO to inactivate liver MAT. Fig. 6 shows the results
obtained with the mutant enzyme R363K; similar results were observed
with the mutant enzymes D355E and G359S (data not shown).
Incubation of WT liver MAT for 10 min with 50 µM
peroxynitrite also induced the inactivation of the enzyme (Fig.
7). As expected, incubation of WT liver
MAT with peroxynitrite did not result in the S-nitrosylation
of the enzyme (data not shown). Whereas replacement of cysteine 121 by
serine prevented the ability of peroxynitrite to inactivate liver MAT,
replacement of aspartic acid 355 or arginine 363 by serine, or
replacement of both arginine 357 and arginine 363 by serine had no
effect on the inactivation of the enzyme by peroxynitrite (Fig. 7).
These results indicate that whereas arginine 357, arginine 363, and
aspartic acid 355 are needed for transnitrosation of cysteine 121, these residues are unneeded for the interaction of this thiol with
peroxynitrite. Moreover, these results provide further evidence that in
these mutants cysteine 121 is not buried in the protein.
Our results indicate that NO or related molecules can regulate
AdoMet content in hepatocytes via reversible NO-mediated
S-nitrosylation and inactivation of hepatic MAT. An increase
in the hepatic levels of NO induces a rapid S-nitrosylation
and inactivation of hepatic MAT (10), which leads to a rapid depletion
of the AdoMet content. Conversely, the elimination of the NO source
leads to the rapid denitrosylation and activation of hepatic MAT (10)
and to the rapid increase of the AdoMet content. This agrees with the
observation that the half-life of hepatic AdoMet is only about 5 min
(6). Further, our results indicate that a moderate increase in the hepatocyte levels of NO, as observed during incubation with 1 mM EGSNO, is sufficient to have a significant effect on
AdoMet content. We have previously established (9) that in rat
hepatocytes kept under low oxygen levels MAT is inactivated. Further we
also found that in rat hepatocytes hypoxia induced the expression of NO
synthase and that the inactivation of MAT during hypoxia was prevented
by the NO synthase inhibitor
NG-monomethyl-L-arginine methyl
ester (9). Elevated levels of NO or related molecules may contribute to
the abnormal metabolism of methionine in patients with liver cirrhosis.
Hypermethioninemia, reduced hepatic MAT activity, and decreased content
of liver AdoMet have been detected in patients with liver cirrhosis
and/or in experimental models of chronic liver injury (23-28). In
patients with liver cirrhosis and in experimental models of chronic
liver disorders serum levels of NOx are elevated (29, 30). The serum NOx levels in patients with liver cirrhosis have been reported to
increase up to 10-fold (from 3 µM in control subjects to
34 µM in cirrhosis) (30). Although it is not known what
may be the concentration of NOx in the cirrhotic liver, the present
results indicate that 50 µM GSNO inactivate liver MAT by
about 60%. These data are evidence for a pathophysiological
interruption of AdoMet metabolism by NO.
NO inhibits many enzymes by S-nitrosylation of active site
and regulatory thiols (31). It is not yet known, however, what structural factors govern the modification of a specific protein cysteine residue by NO. An acid-base-catalyzed SNO/SH exchange reaction, where the target cysteine residue is located next to basic
and acid amino acids, has been recently proposed (11). The NO-mediated
inactivation of hepatic MAT appears to occur through S-nitrosylation of the residue of cysteine 121 of the enzyme
(8, 10). Our results indicate that the substitution of cysteine 121 by
serine prevents MAT S-nitrosylation and inactivation by GSNO. Further we show that there is a close inverse correlation between
the extent of S-nitrosylation and loss of activity. Cysteine 121 is localized at a "flexible loop" over the active site cleft of
MAT (32-34). This loop can adopt two different conformations, open and
closed, and it has been proposed that in the closed conformation prevents the entrance of the substrates to the active site (34). Cysteine 121 is not essential for activity because the substitution of
this amino acid residue by serine has no effect on MAT activity (15).
The present results suggest that formation of a SNO group in cysteine
121 induces a conformational change in the "flexible loop" making
less accessible the active site of the enzyme for the substrates,
probably by switching the loop into the closed conformation.
Replacement of the acidic amino acid (Asp-355) or of any of the two
basic amino acids (Arg-357, Arg-363) localized in the proximity of
cysteine 121 by serine markedly reduced the capacity of GSNO to
S-nitrosylate and inactivate liver MAT. Further, our results
indicate that GSNO-induced MAT S-nitrosylation and inactivation are greatly prevented in a double mutant enzyme where arginine 357 and arginine 363 were both replaced by serine. When the
changes did not involve loss of charge (arginine 363 by lysine and
aspartic acid 355 by glutamic acid) these mutations had no effect on
the S-nitrosylation and inactivation of the mutant enzymes by GSNO. Our results also indicate that the change of a non-charged amino acid (glycine 359) in the vicinity of cysteine 121 by serine did
not affect S-nitrosylation and inactivation of the mutant enzyme by GSNO. We also show that peroxynitrite-induced MAT
inactivation involves the reaction of this oxidizing agent with
cysteine 121. In this case, however, replacement of arginine 357, arginine 363, or aspartic acid 355 by serine did not affect
peroxynitrite-induced MAT inactivation. These results indicate that the
decreased GSNO-mediated MAT S-nitrosylation and inactivation
seen following replacement of acidic and basic residues in the vicinity
of cysteine 121 with serine are because of loss of charge and are not
because of structural changes that alter the position or otherwise
protect the thiol. One possible explanation of these findings is that
the function of the guanidino groups of arginine 357 and arginine 363 may be to facilitate the deprotonation of the sulfur group of cysteine 121 (Fig. 8). This will increase the
nucleophilicity of cysteine 121 (by lowering its
pKa) and consequently facilitate the nitrosylation
of its sulfur group. The function of the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of EGSNO on AdoMet content in isolated
rat hepatocytes. Isolated rat hepatocytes were incubated in the
absence ( ) or in the presence (
) of 5 mM EGSNO. After
incubation with EGSNO for 55 min, The NO donor was removed from the
medium by centrifugation, and after washing the cells twice, the
hepatocytes were resuspended in the absence (
) or presence (
) of
5 mM EGSNO. AdoMet content was determined as described
under "Experimental Procedures" at the times indicated in the
figure. Values are expressed as means ± S.E. of three independent
experiments in duplicate.
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Fig. 2.
Effect of EGSNO on AdoMet content and NOx
levels. Hepatocytes were incubated with various concentrations of
EGSNO. Cytosolic AdoMet ( ) and NOx (
) content were determined
after 20 min of incubation with the NO donor as described under
"Experimental Procedures." Values are expressed as means ± S.E. of three independent experiments in duplicate.
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Fig. 3.
Dose-dependent
S-nitrosylation of WT and mutant rat liver MAT by
GSNO. Recombinant MAT purified from Escherichia coli
transformed with WT and mutant cDNA were incubated in the presence
of various concentrations of GSNO for 10 min. The formation of SNO
groups was determined as described under "Experimental Procedures."
Values are expressed as means ± S.E. of three experiments in
triplicate using different preparations of the enzyme. , WT;
,
C121S;
, R363S/R357S;
, R357S;
, R363S;
, D355S;
,
R363K. Mutants are identified by a number that indicates the amino acid
residue in the sequence of the enzyme that has been replaced.
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Fig. 4.
Correlation between extent of MAT
S-nitrosylation and loss of activity. MAT
purified from E. coli transformed with WT mutant cDNA
was incubated in the presence of various concentrations of GSNO (5-100
µM) for 10 min at 37 °C. The enzyme activity and the
formation of SNO groups were then determined as described under
"Experimental Procedures." MAT activity is expressed as the percent
of activity remaining after treatment with GSNO. Values are the
mean ± S.E. of three different enzyme preparations
(r = 0.95; p < 0.001).
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Fig. 5.
A model structure of rat liver MAT. The
structural detail of the microenvironment of cysteine 121 is based on a
model structure for the rat liver MAT subunit published previously (32)
using the data available for the x-ray crystal structure of E. coli MAT (33, 34). This figure was prepared with Ras Mol (35). The
position of cysteine 121 (C121), arginine 357 (R357), arginine 363 (R363), and aspartic acid
355 (D355) are indicated. According to this model, the
distances between the SH group of cysteine 121 and the guanidino groups
of arginine 357 and arginine 363 are 3.3 and 4.4 Å, respectively. The
distance between the SH group of cysteine 121 and the -COOH of
aspartic acid 355 is 4.9 Å.
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Fig. 6.
Inactivation of WT and mutant rat liver MAT
by GSNO. MAT purified from E. coli transformed with WT
and mutant cDNA were incubated in the absence or presence of 50 µM GSNO for 10 min. The enzyme activity was then measured
as described under "Experimental Procedures." Results are expressed
as the percent of activity remaining after treatment with GSNO for each
individual mutant. Values are expressed as mean ± S.E. of three
different preparations. Mutants are identified by a number that
indicates the amino acid residue in the sequence of the enzyme that has
been replaced.
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Fig. 7.
Inactivation of WT and mutant rat liver MAT
by peroxynitrite. MAT purified from E. coli transformed
with WT and mutant cDNA were incubated in the absence or presence
of 50 µM peroxynitrite for 10 min. The enzyme activity
was then measured as described under "Experimental Procedures."
Results are expressed as the percent of activity remaining after
treatment with peroxynitrite for each individual mutant. Values are
expressed as mean ± S.E. of three different preparations. Mutants
are identified by a number that indicates the amino acid residue in the
sequence of the enzyme that has been replaced.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-COOH group of aspartic
acid 355 may be to facilitate the protonation of GSNO and, accordingly,
facilitate the donation of its NO group. These results provide the
first experimental evidence that, as proposed by Stamler et
al. (11), the S-nitrosylation of protein thiol residues
is governed by the basic and acidic amino acids surrounding the target
thiol. Recognition of this topology is likely to prove useful in
identifying new targets of protein S-nitrosylation.
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Fig. 8.
A model for the reaction of cysteine 121 with
GSNO. Whereas the guanidino moiety of arginines 357 and 363 facilitates the deprotonation of the sulfur group of cysteine 121 and
thus increases its nucleophilicity, the -COOH group of aspartic acid
355 facilitates the protonation of GSNO and consequently enhances the
donation of its NO group.
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ACKNOWLEDGEMENTS |
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We thank Prof. Manuel Martín Lomas and Dr. Dacil Zurita for the synthesis of EGSH.
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FOOTNOTES |
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* This work was supported by the Plan Nacional de I+D (SAF 98/132), Europharma, and Knoll.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.
Fellow of the Ministerio de Educación y Cultura.
§ Fellow of the University of Navarra.
¶ Fellow of the Instituto de Cooperación Iberoamericana.
To whom correspondence should be addressed. Tel.: 34-948 42 56 78; Fax: 34-948 42 56 77; E-mail: jmmato{at}unav.es.
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ABBREVIATIONS |
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The abbreviations used are: AdoMet, S-adenosylmethionine; NO, nitric oxide; MAT, methionine adenosyltransferase; SNO, nitrosothiol; EGSH, glutathione monoethyl ester; GSNO, S-nitrosoglutathione; EGSNO, S-nitrosoglutathione monoethyl ester; WT, wild type; NOx, nitrite and nitrate.
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