From the Institute of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
Received for publication, September 5, 2002, and in revised form, November 15, 2002
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ABSTRACT |
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The endogenous nitric oxide synthase inhibitors
L-N The free radical NO acts as a signaling molecule in various
tissues. In neuronal tissue it functions as a neurotransmitter that, as
a gas molecule, is able to freely permeate through cell membranes. NO
binding to the heme moiety of soluble guanylate cyclase activates this
enzyme, leading to the generation of the second messenger, cyclic GMP
(1). Apart from this signaling pathway, NO plays an important role in a
number of biological processes, including the regulation of protein
function through the nitrosation of sulfur atoms of Cys residues (2).
Well characterized examples are those of caspase-3 (3) or the
N-methyl-D-aspartate (NMDA)1 receptor (4). In most
examples of Cys-S-nitrosylation, the target protein is
inhibited. NO is produced by at least three isoforms of nitric oxide
synthase (NOS) of which nNOS occurs predominantly in neuronal tissues
(5). The activity of this isoform is mainly regulated by
Ca2+ (6). Several other regulatory mechanisms for nNOS,
including its inhibition by phosphorylation, have also been described
(6). It is well established, moreover, that the two L-Arg
derivatives, L-N Increased levels of MMA and ADMA in plasma and urine were found in
neuronal diseases characterized by low NO levels such as schizophrenia
(12) as well as diseases linked to vascular dysfunction, e.g. uremia, atherosclerosis, hypercholesterolemia, diabetes
mellitus, hypertension, and homocysteinemia (13). Conversely, NO
overproduction is apparently responsible for migraine (14). In this
case, the observed increase in expression of various enzymes including
DDAH has been linked to an imbalance in NOS inhibitors (15).
In mammals, two isoforms of DDAH, DDAH-1 and DDAH-2, exist (16).
Whereas DDAH-2 is mainly expressed in the heart, kidney, and placenta,
DDAH-1 is predominantly expressed in the brain (16). The specific role
for DDAH-1 in the regulation of nNOS has been suggested based on recent
studies showing that both nNOS and DDAH-1 are up-regulated in injured
neurons (17). The characterization of DDAH-1 from bovine brain revealed
a monomeric 31.2-kDa protein containing one non-catalytic Zn(II)
(18-20). Activity measurements of native and metal-free DDAH-1 showed
that the endogenously bound Zn(II) inhibits the enzyme. Extended x-ray
absorption fine structure (EXAFS) studies suggested that Zn(II) is
tetrahedrally coordinated through two sulfur (Cys) ligands and two
nitrogen (or oxygen) ligands (20). However, Zn(II)-containing DDAH-1
could be fully or partially activated by various concentrations of
phosphate, imidazole, histidine, and histamine, a process accompanied
by the release of Zn(II). Based on this and the apparent Zn(II)
dissociation constant in the nanomolar range, a regulatory role for
Zn(II) has been suggested (19, 20). Increased levels of intracellular Zn(II) under oxidative or nitrosative stress conditions are well documented (21-23). This may account for the reduction of DDAH-1 activity and the presence of elevated ADMA levels observed in cell
culture studies under these conditions (10). Alternatively, because
DDAH is a cysteine hydrolase, the enzyme inhibition through its
nitrosation is also likely. Upon submission of the present work, the
in vitro inhibition of prokaryotic DDAH by NO donors has
been reported. Moreover, in endothelial cell cultures heterologously expressed DDAH-2 was found S-nitrosylated following the
cytokine induced expression of inducible NOS. In both instances, DDAH
nitrosation was detected by an anti-S-nitrosocysteine
antibody (24).
The present work was conducted with the aim of gaining insights into
the effect of NO on the structure and activity of mammalian DDAH-1
in vitro. The nitrosation reaction was studied with the Zn(II)-containing and Zn(II)-free enzymes. DDAH-1 nitrosation was
followed by electrospray ionization mass spectrometry (ESI-MS), electronic absorption spectroscopy, fluorometric SNO quantification, and measurements of enzymatic activity. The results showed that whereas
the holo-form is resistant to nitrosation, two of five Cys residues of
the apo-form were modified. To examine the specificity of the reaction,
the amino acid sequence of DDAH-1 was determined, and the tryptic
peptides of the NO modified enzyme were analyzed by ESI-MS. The results
allowed the conclusion that the formation of Cys-S-NO is
specific and that Zn(II) protects the enzyme against NO modification.
Homology modeling revealed that the close proximity of charged residues
in the tertiary structure of DDAH-1 might be responsible for the
specific Cys-S-nitrosylation.
MMA·HOAc and sodium
2-(N,N-dimethylamino)-diazenolate-2-oxide (DEA
NONOate) were purchased from Alexis, and
Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP·HCl) was
purchased from Pierce. Trypsin from bovine pancreas (sequencing grade)
was obtained from Roche Molecular Biochemicals, and the endoproteinase
Glu-C from the Staphylococcus aureus strain V8 was purchased
from Sigma. Dialysis tubing (cut off, 12-14 kDa; Spectrum) was
rendered metal-free as described (25). DDAH-1 was purified from bovine
brain using the previously reported procedure (20). The protein
homogeneity was checked by SDS-PAGE and ESI-MS.
Metal and Protein Quantification--
The zinc concentration was
determined by atomic absorption spectroscopy (IL Video 12), and that of
DDAH-1 was determined by absorption spectroscopy (Cary 3, Varian) using
the molar extinction coefficient Activity Measurements--
The product of the enzymatic reaction
(L-Cit) was determined by our previously reported
colorimetric method in 96-well microtiter plates (26). If not otherwise
stated, upon substrate addition (6.7 mM MMA) the samples
were incubated for 30 min at 37 °C. DDAH-1 concentrations in the
range of 0.5-1 µM were used. As only apo-DDAH-1 is
active, in some instances this form was prepared by in situ incubation of the native enzyme in 250 mM imidazole/HCl, pH
7.4 (20). All activity measurements were performed in triplicate. One
unit of DDAH-1 is defined as the amount of enzyme that produces 1 µmol of L-Cit per minute under the conditions given.
Large Scale Reintroduction of Zn(II) into DDAH-1--
Isolated
DDAH-1 from bovine brain contained between 0.95 and 0.85 mol
equivalents of Zn(II). To generate a fully metal-loaded DDAH-1, Zn(II)
was introduced into the protein structure through dialysis after
protein reduction. Briefly, to 10 ml of freshly isolated DDAH-1 (~11
µM), 20 µl of 0.5 mM TCEP was added. The solution was subsequently dialyzed against two changes (every 2 h)
of 1 liter of 10 mM Tris/HCl, 100 mM KCl, pH
7.8, at 4 °C. In the first dialysis step 10 mM
2-mercaptoethanol was present. Finally, the solution was dialyzed
overnight against 1 liter of 10 mM Tris/HCl, 100 mM KCl, and 50 µM ZnSO4, pH 7.8. In all dialysis steps, the outer solution was steadily bubbled with
argon. To remove unbound Zn(II), the dialyzed solution was desalted
over a Superdex G75 HR16/60 gel permeation column (Amersham
Biosciences) equilibrated in 10 mM MES/NaOH and 100 mM KCl, pH 6.2. DDAH-1-containing fractions were combined
and concentrated using Centriprep-10 (Millipore).
Generation of Zn(II)-free DDAH-1--
The preparation of the
apo-enzyme was performed by dialysis. All dialysis steps were carried
out at 4 °C, and the outer solutions were steadily bubbled with
argon. Buffers were rendered metal-free as described previously (19).
To remove Zn(II) from native DDAH-1, 200 µl of protein sample (~50
µM) were dialyzed against 100 ml of metal-free 100 mM imidazole/HCl and 10 mM DTT, pH 6.8. The dialysis buffer was changed four times (every 2 h), and the
dialysis was completed overnight. To remove imidazole and DTT, the
protein was dialyzed against two changes of 100 ml of metal-free 10 mM MES/NaOH and 100 mM KCl, pH 6.2.
Inactivation of DDAH-1 through NO--
To monitor DDAH-1
activity as a function of the DEA NONOate concentration in the presence
and absence of Zn(II), both holo- and apo-DDAH-1 (8.7 µM)
were tested either in 250 mM MES/NaOH, 20% (v/v) glycerol,
pH 6.4, or 250 mM imidazole/HCl, 20% (v/v) glycerol, pH
6.4. To the protein samples, DEA NONOate was added to a final
concentration of 0, 0.2, 0.5, 1, 2, 5, or 10 mM, and the
solution mixture was incubated for 20 min at room temperature. Subsequently, samples of 3 µl were removed and added to 57 µl of
250 mM imidazole/HCl and 7 mM MMA, pH 7.4. Finally, the mixtures were incubated for 30 min at 37 °C, and their
L-Cit concentrations were determined.
To monitor the time dependence of DDAH-1 activity in the presence of
constant NO donor concentration, two samples of holo-DDAH-1 were
incubated in 250 mM imidazole/HCl, pH 6.4, containing
either 1 mM or 5 mM DEA NONOate. Aliquots were
taken after 0, 10, 20, 40, or 90 min, and their residual enzymatic
activities were determined.
Correlation of Cys-S-Nitrosylation with DDAH-1 Activity--
Six
samples of 10 µM apo-DDAH-1 in 250 mM
imidazole/HCl, pH 6.4, were incubated with 0, 0.2, 0.7, 5, 10, or 20 mM DEA NONOate for 30 min at 37 °C. Aliquots of 2 µl
of each sample were placed into a polystyrene 96-well microtiter plate
and diluted with 50 µl of 250 mM imidazole/HCl, pH 6.4. Upon the addition of 5 µl of 80 mM MMA, the plate was
incubated for 30 min at 37 °C. Afterward, the L-Cit
concentration was determined.
In parallel to the activity measurements, the number of
Cys-S-NOs was quantified. For this purpose the fluorometric
method of Park and Kostka (27) was adapted for use in 96-well
microtiter plates. Briefly, another 2-µl aliquot from the
initial DEA NONOate incubation were added to 98 µl of 1% (w/v)
ammonium sulfamate in 0.25 mM HCl placed in a white 96-well
microtiter plate. The probes were incubated for 10 min at room
temperature. In the next step, 10 µl of 0.05 mg/ml
2,3-diaminonaphthalene in 0.62 M HCl and 5 µl
of 4 mM HgCl2 in 0.25 mM HCl were
added to each well. The probes were incubated for 10 min at room
temperature. Samples treated with 5 µl of 50 mM
Na2-EDTA in 0.25 mM HCl instead of HgCl2 were taken as blanks. Finally, to each sample 5 µl
of 2.8 M NaOH was added. After 15 min at room temperature
the fluorescence at 430 nm (excitation at 360 nm) was determined using
a microtiter plate reader (HTS 7000 Plus, PerkinElmer Life Sciences). A
stock solution of NaNO2 was calibrated photometrically at
210 nm employing the molar extinction coefficient Spectroscopic Characterization of Nitrosated DDAH-1--
For the
spectroscopic characterization of nitrosated DDAH-1, 450 µl of 26 µM DDAH-1 in 250 mM imidazole/HCl, pH 6.4, were titrated with DEA NONOate. The titration was performed with 1-µl aliquots of 100 mM DEA NONOate in 10 mM NaOH.
Spectra were recorded between 300 and 500 nm (Cary 3, Varian Inc.) 10 min after NO donor addition. The sample was titrated until a maximum
was reached (about 10 mM DEA NONOate added in total).
Mass Spectrometric Analysis of Nitrosated DDAH-1--
20 µl of
12 µM apo-DDAH-1 in 20 mM
NH4OAc/HOAc, pH 6.4, were incubated with 0, 5, or 20 mM DEA NONOate for 20 min at 37 °C. Before injection,
samples were desalted using C4-ZipTips (Millipore) from
which proteins were eluted with 10 µl of 78:21:1 (v/v/v) CH3OH/H2O/HCOOH. ESI mass spectra of 5 µl of
this solution were acquired on an API III+ triple
quadrupole instrument (AB/MDS Sciex, Toronto, Canada). The following
set of parameters was applied: ionization voltage, 4800 V; orifice
voltage, 80 V; mass range, m/z 700-1700; step size, 0.15 Da; scan duration, 5 s.
Amino Acid Sequence Determination of DDAH-1--
We have already
determined 20% of the amino acid sequence of bovine brain DDAH-1 using
peptides of a tryptic digest (19). This approach was expanded in the
present studies using peptides from digests with trypsin and
endoproteinase Glu-C. The resulting peptides were separated by HPLC and
analyzed using a combination of MS/MS and Edman peptide sequencing. The
tryptic digest was performed as described (19). The second digest was
done with one-twentieth of endoproteinase Glu-C in 25 mM
NH4HCO3/NH3, 0.5 M
guanidinium chloride, pH 7.8, for 2 h at 25 °C. The sequenced peptides were aligned according to the mammalian DDAH-1 sequences available to date.
Position of Cys-S-NO in DDAH-1 Amino Acid Sequence--
10
µM DDAH-1 was incubated in 260 µl of 250 mM
imidazole/HCl, pH 6.4, with 20 mM DEA NONOate for 30 min at
37 °C. The sample was washed extensively with 300 µl of 0.1%
(v/v) TFA in a Microcon-10 (Millipore). After a final washing step with
100 mM Tris/HCl and 10% (v/v) CH3CN (pH 8.0)
to a final volume of 150 µl, 0.2 mol equivalents of trypsin were
added, and the sample was incubated for 2 h at 37 °C.
100 µl of the digest were mixed with 1 µl of TFA and applied to a
reversed phase HPLC (Vydac MS-C18, 1 × 254 mm). The
flow rate was 50 µl min Determination of a Model Structure for Bovine DDAH-1--
The
model structure of the bovine DDAH-1 was calculated using the program
3D-PSSM V2.6.0 (www.sbg.bio.ic.ac.uk/~3dpssm/) (29). The
bovine DDAH-1 sequence aligned best against Pseudomonas
aeruginosa DDAH (PDB #1H70). A further refinement was performed
using the program Swiss-PdbViewer V3.7 (30), placing the amino acid sequence of bovine DDAH-1 (this work) and the x-ray diffraction coordinates of P. aeruginosa DDAH (31). A final
energy minimization was done with the GROMOS96 implementation of the
Swiss-PdbViewer using 50 steps of steepest descent and 100 steps of
conjugated gradients.
Bovine brain DDAH-1 used in this study contained ~0.95 mol
equivalents of Zn(II). Previously, we could show that the bound metal
inhibits the enzyme and that only the apo-enzyme is active. Zn(II) is
bound with an apparent dissociation constant of 4.2 nM
(20). Metal removal by strong and rather bulky metal chelators like
EDTA or 1,10-phenanthroline takes place very slowly. However, Zn(II)
can be dissociated from the protein rather easily in the presence of
high concentrations of imidazole/HCl or phosphate buffer (100-500
mM), suggesting that the metal ion is buried in the protein
structure. In other common buffers such as MES/NaOH, HEPES/NaOH,
Tris/HCl, and TEA/HCl, no metal release occurs (20).
Inhibition of Apo-DDAH-1 with DEA NONOate--
The influence of
varying concentrations of the NO donor DEA NONOate on the activity of
holo-DDAH-1 was examined in either 250 mM MES/NaOH or 250 mM imidazole/HCl at pH 6.4. Upon enzyme incubation in both
buffers with DEA NONOate, the protein was transferred to 250 mM imidazole/HCl, pH 7.4. Note that in the latter buffer the activity of the in situ generated apo-form was measured
(20) (see above). It appeared that the enzyme pre-incubated in
imidazole/HCl was dramatically inhibited by NO, whereas its inhibition
in MES/NaOH was only minor (Fig.
1A). In addition, apo-DDAH-1
was also generated through dialysis of the holo-enzyme as described
(19) and incubated with DEA NONOate both in 250 mM MES/NaOH
and 250 mM imidazole/HCl. In both buffers, the effect of NO
on the enzymatic activity was similar to that observed with holo-DDAH-1
in 250 mM imidazole/HCl (data not shown). These results
allow the conclusion that whereas apo-DDAH-1 is inhibited by NO, the
presence of Zn(II) in the protein structure protects the enzyme against
nitrosation. To further characterize the nitrosated DDAH-1, the
experimental conditions under which the major part of the protein is
nitrosated were sought. For this purpose, in situ generated
apo-DDAH-1 was incubated with 1 or 5 mM DEA NONOate, and
the changes in enzymatic activity followed as a function of time (Fig.
1B). In both cases, a hyperbolic decrease of activity was
discerned. The ~75% enzyme inhibition seen after 20 min of sample
incubation with 5 mM NO donor (Fig. 1B) is very similar to that reached with 10 mM NO donor in the same
time (Fig. 1A).
Characterization of the Product of DDAH-1 Nitrosation--
Protein
nitrosation gives rise to various protein modifications. Besides the
formation of Cys-S-NO, which has been described for many
proteins (2, 32), the formation of Tyr-NO2 (33), Cys-Cys
(34), Cys-S-OH, Cys-S-O2H, and
Cys-S-O3H were also reported (35-37). To
characterize the species formed in the case of nitrosated DDAH-1, its
absorption spectrum between 300-500 nm was recorded (Fig.
2A). The spectrum reveals a
maximum at 341 nm being characteristic for the
nO Quantification of NO-modified Cys Residues in DDAH-1--
Using
electronic absorption spectroscopy and ESI-MS, the nitrosation product
of the reaction of DEA NONOate with DDAH-1 could be unambiguously
characterized (see above). However, neither of these two methods
provides quantitative results. Thus, depending on the protein, the
molar absorption coefficients per Cys-S-NO substantially
differ (39). In addition, the ordinary ESI-MS spectra allow only a
qualitative analysis. For the quantification of Cys-S-NO we
have employed the fluorescence detection of 1-[H]-naphtotriazol, generated in the reaction of 2,3-diaminonaphthaline with
Hg(II)-released NO. The method described by Park and Kostka (27) was
modified for the use in 96-well microtiter plates. The degree of
Cys-S-NO modification as a function of increasing DEA
NONOate concentrations (between 0-20 mM) was examined. The
results were correlated with the residual enzymatic activity. Fig.
3 reveals that the absence of enzymatic
activity above 10 mM DEA NONOate is paralleled by the
modification of ~2.5 Cys residues. Taken together, it would appear
that two of five Cys residues in DDAH-1 are preferentially S-nitrosylated.
Determination of the Amino Acid Sequence of Bovine Brain
DDAH-1--
We have recently developed a fast isolation procedure
yielding 1-1.5 mg of DDAH-1 from 100 g of bovine brain tissue and
have determined its partial amino acid sequence (20%) (19, 20). Because our search for DDAH-1 cDNA in a commercial Localization of Cys-S-NO in Bovine DDAH-1--
Cys-S-NO
derivatives are rather unstable at alkaline pH values (39). Therefore,
the tryptic digest for the detection of the Cys-S-NO within
the DDAH-1 sequence was performed at pH 8.0 using a high trypsin to
DDAH-1 ratio and a short incubation time (2 h) (42). A comparison of
the peptide maps of the untreated and nitrosated DDAH-1 revealed two
Cys residues containing peptides Leu-211 to Lys-229 and Val-266 to
Lys-280 in which a mass difference of 29 Da was found (Table
I). The amount of
S-nitrosylated species was estimated from the ratio of the
counted ions in the LC-MS experiment of the nitrosated protein (Table
I). In a third peptide (Leu-57 to Arg-97) containing two Cys residues
(Fig. 4), only a minor nitrosation of each single Cys was found. The
two mono-nitrosated peptides eluted in two different chromatographic
peaks (Table I). No double nitrosation of this peptide was
detected.
The assignment of the peptides containing S-nitrosylated
Cys-221 and Cys-273 was confirmed by MS/MS analysis. Even at relatively low collision energies, an almost complete homolytic cleavage of the
S-NO bond occurred in agreement with previous studies (43). At higher
collision energies, the peptide ions resulting from a loss of the NO
radical further fragmented, giving rise to
sequence-dependent fragment ions of the denitrosated
peptides. The NH2- and COOH-terminal ions of the a, b, and
y series were accompanied by relatively abundant, uncommon b-1 and y-1
ions (a discussion of the nomenclature used in identifying fragment
ions can be found in Ref. 44). The b-1 and y-1 ions appear to contain
free radicals, which originate from cleavage of the S-NO bond.
Homology Model of DDAH-1--
The crystal structure of
P. aeruginosa DDAH has recently been determined
(31). Because bovine DDAH-1 and the P. aeruginosa DDAH share
an amino acid sequence identity of 30%, it is possible to obtain a
model structure for bovine DDAH-1 using the crystallographic data
available for the P. aeruginosa protein. The model building was based on the sequence alignment shown in Fig. 4, which was obtained
from the secondary structure prediction by PSI-Pred
(www.globin.bio.warwick.ac.uk/psipred/) included in the program 3D-PSSM
(29). Fig. 5A shows the
superposition of the prokaryotic DDAH crystal structure
(white) with the DDAH-1 model structure (blue).
The root mean square deviation of 1.71 Å between both structures
indicates a high significance of the model. The fold of the prokaryotic
DDAH consists of five modules of more or less degenerated
In the present work we have demonstrated that only apo-DDAH-1
reacts with NO, leading to enzyme inhibition (Fig. 1). The reaction product could be identified as SNO-DDAH-1. A fluorometric
quantification of Cys-S-NO revealed the nitrosation of two
of five Cys residues (Fig. 3). To answer the question as to the
specificity of the Cys-S-NO formation, the amino acid
sequence of bovine brain DDAH-1 was determined, and the nitrosated
apo-enzyme was subjected to a tryptic digest. From the ratio of the
counted ions of the nitrosated and the non-nitrosated peptides in an
LC-MS experiment, semi-quantitative results were obtained. The product
analysis showed the presence of two major
Cys-S-NO-containing peptides (Table I), with Cys-221 and
Cys-273 being the major S-nitrosylated residues. A slight nitrosation of the third tryptic peptide of DDAH-1 (see Table I)
presumably represents a side product of the reaction or a product of NO
interchange with Cys-S-NO-containing peptides upon the
tryptic digest. Thus, the results suggest that the enzyme nitrosation
is specific. Because Cys-273 is the active site residue of DDAH-1 (Fig.
4), the inhibition of the enzymatic activity is due to its
S-nitrosylation. However, both the correlation between the
number of Cys-S-NO residues and the enzymatic activity (Fig. 3) and the LC-MS-experiments (Table I) suggest that Cys-221 is S-nitrosylated to the same degree as Cys-273.
The specific Cys-S-nitrosylation is surprising, because in
the primary structure of bovine DDAH-1 the sequence motif
XC(D/E) for Cys-S-nitrosylation (2, 32), where
X usually stands for basic or acidic amino acids, is absent.
A growing number of proteins have been recognized as being regulated
through the S-nitrosylation of specific Cys residues (2). In
many examples, including caspase-3 and methionine adenosyl transferase,
the preference of a specific Cys for S-nitrosylation seems
to be achieved through a close spatial contact resembling the continues
sequence motif mentioned above (46, 47). To gain an insight into the
structural features of bovine DDAH-1 responsible for the specific
Cys-S-nitrosylation, a homology model of this enzyme was
generated (Fig. 5, A and B). Modeling studies
based on the structure of P. aeruginosa DDAH have suggested
the presence of five similar To evaluate whether structural properties of bovine DDAH-1 are also
important for the activation of both Cys-221 and Cys-273 for
Cys-S-nitrosylation, the structural model was examined
further. Its closer inspection revealed that the S The Cys-S-nitrosylation of enzymes is often accompanied by a
reversible loss of activity (2). The enzyme activity of DDAH-1 is
inhibited with IC50 The reversibility of this reaction was tested by incubation of
SNO-DDAH-1 with 5 mM 2-mercaptoethanol, DTT, or
glutathione. However, in contrast to a number of reports on other
nitrosated proteins/enzymes where such a treatment restored the
enzymatic activity, our attempts to restore the enzymatic activity of
nitrosated DDAH-1 remained unsuccessful (data not shown). Because the
removal of Cys-S-bound NO from proteins is likely to occur
through trans-nitrosylation, we ascribe this effect to the
inaccessibility of the nitrosylated Cys-273 and Cys-221 residues to
these reagents. In the model of DDAH-1, both residues are buried in the
protein structure (Fig. 5B). It should be noted that also in
the case of caspase-3 the reversibility of the
Cys-S-nitrosylation is partial and requires a rather high
DTT concentration (47).
Apart from the enzyme nitrosation, DDAH-1 is also inhibited by Zn(II)
ions with an apparent binding constant lying in the range of
intracellular "free" Zn(II) concentrations (20). Based on activity
measurements of DDAH-1 in brain tissue homogenate, it would appear that
~30% of the enzyme is present in the Zn(II)-bound form (20). In many
Zn(II)-containing proteins such as metallothioneins, zinc finger
proteins, and liver alcohol dehydrogenase, NO binds preferentially to
Cys-S ligands, thereby displacing bound Zn(II) (51-54).
However, our data demonstrate that for DDAH-1 this is not the case.
Because the bound Zn(II) on the one hand inhibits the enzyme and on the
other hand protects the two Cys residues against
S-nitrosylation, we propose that both Cys-221 and Cys-273 also act as the Zn(II) ligands. Evidence for the presence of thiolate ligands in the Zn(II) coordination sphere came from the EXAFS studies,
which revealed that the metal ion is tetrahedrally coordinated by two
sulfur ligands (Cys) and two lighter ligands (nitrogen or
oxygen) (20). In the structural model of DDAH-1, the distance between
the S The reduction of DDAH-1 activity and the presence of elevated ADMA
levels were observed in cell culture studies under oxidative stress
conditions (10). This observation was paralleled by a decrease in NO
production (55, 56), suggesting that under these conditions
intracellular DDAH activity would be substantially inhibited. In very
recent endothelial cell culture studies, heterologously expressed
DDAH-2 was found S-nitrosylated following the
cytokine-induced expression of inducible NOS. The enzyme nitrosation
was established by means of an anti-S-nitrosocysteine
antibody (24). Independently, in vitro
S-nitrosylation of one (Cys-249) of a total of five Cys residues of structurally well characterized bacterial DDAH has also
been shown. In the present work, S-nitrosylation of two Cys residues of DDAH-1 (Cys-221 and Cys-273) has been demonstrated. The
differences in the primary structures between bovine DDAH-1 and
P. aeruginosa DDAH (30% identity) are most likely the
reason for this effect. An inhibition of protein function by both
Zn(II) and NO has already been reported, e.g. in the case of
caspase-3 (47) and the NMDA receptor (57). Whereas for the NMDA
receptor the residues involved in Zn(II) binding are to date not known, for caspase-3 the active site Cys is clearly the residue involved in
the binding of both Zn(II) and NO (47). Although both agents are
involved in caspase-3 regulation, details of this mechanism are
currently unknown. In view of our studies, both Zn(II) and NO would be
involved in the regulation of DDAH-1, which, in turn, through the
variation of MMA and ADMA levels would regulate NOS activity. Thus,
more biological and structural studies are needed to understand the
cellular regulation of DDAH-1.
-methylarginine and
L-N
,N
-dimethylarginine
are catabolized by the enzyme dimethylargininase. Dimethylargininase-1
from bovine brain contains one tightly bound Zn(II) coordinated by two
cysteine sulfur and two lighter ligands. Activity measurements showed
that only the apo-enzyme is active and that the holo-enzyme is
activated by zinc removal. In this work, the effect of NO on
dimethylargininase-1 structure and its activity was investigated using
2-(N,N-dimethylamino)-diazenolate-2-oxide as an
NO source. The results showed that whereas the holo-form was resistant
to S-nitrosylation, the apo-form could be modified. The
results of absorption spectroscopy, mass spectrometry, and fluorometric
S-NO quantification revealed that two of five cysteine residues reacted
with NO yielding cysteine-S-NO. The modification reaction
is specific, because by liquid chromatography/mass spectrometry experiments of digested S-NO-dimethylargininase-1, cysteines 221 and
273 could be identified as cysteine-NO. Because Zn(II) protects the
enzyme against nitrosation, it is suggested that both cysteines are
involved in metal binding. However, specific cysteine-S-NO formation occurred in the absence of a characteristic sequence motif.
Based on a structural model of dimethylargininase-1, the activation of
both cysteines may be accomplished by the close proximity of
charged residues in the tertiary structure of the enzyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-methylarginine
(MMA) and
L-N
,N
-dimethylarginine
(ADMA), act as endogenous competitive inhibitors of NOS (7-10). Their
cytosolic concentrations are regulated by the enzyme
dimethylargininase
(L-N
,N
-dimethylarginine
dimethylaminohydrolase, DDAH, EC 3.5.3.18). This cysteine hydrolase
catabolizes MMA and ADMA to L-citrulline (L-Cit) and CH3NH2 or
(CH3)2NH, respectively (11).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
280 nm = 14420 M
1 cm
1 (19).
210 nm = 5380 M
1 cm
1 (28). The
standard NaNO2 curve was obtained by adding 2 µl of 0, 5, 10, 20, 50, 100, 200, or 500 µM NaNO2 into 98 µl of 0.25 mM HCl, and the samples were treated as
described for the blanks. All measurements were performed in triplicate.
1, and the absorption was
monitored at 215 nm. Eluent A consisted of 0.02% (v/v) TFA, and eluent
B was 80% (v/v) CH3CN and 0.02% (v/v) TFA. The following
gradient program was applied: 0 min 5% B; 5 min 5% B; 65 min 50% B;
75 min 60% B; 80 min 100% B. The eluate was split to allow 10% to
enter the electrospray source (5 µl min
1), the
remainder being diverted for manual fraction collection. ESI mass
spectra were acquired from m/z 450 to 1700 with a
step size of 0.25 Da and a scan duration of 5 s. MS/MS spectra of
the doubly charged S-nitrosylated peptides were recorded in
the collision energy range of 32-42 eV using argon (2.5 × 1014 molecules cm
2) as collision gas.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
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Fig. 1.
Effect of NO on the enzymatic activity of
native DDAH-1. A, dependence of the enzymatic activity
of DDAH-1 on DEA NONOate concentration. Samples were pre-incubated with
DEA NONOate at room temperature for 20 min in 250 mM
MES/NaOH, pH 6.4 ( ), or 250 mM imidazole/HCl, pH 6.4 (
). The presented activity was obtained in 250 mM
imidazole/HCl, pH 7.4, at 37 °C. B, time dependence of
DDAH-1 activity in the presence of 1 mM (
) and 5 mM (
) DEA NONOate in 250 mM imidazole/HCl,
pH 6.4. The presented data were obtained as for panel
A). For details see "Materials and Methods."
* transition of the SNO group (38, 39).
Independent evidence for the presence of Cys-S-NO was
provided by ESI-MS spectra recorded upon the addition of 0, 5, or 20 mM DEA NONOate to apo-DDAH-1 (Fig. 2,
B-D). Besides the major peak of apo-DDAH-1 at
31199 Da (arrow), up to three additional species with masses
of 31228 (a), 31256 (b), and 31285 Da
(c) (Fig. 2, C and D) were observed.
The additional peaks (a-c) with mass differences
of a multiple of 29 Da compared with the unmodified protein suggest the
presence of DDAH-1 species containing one, two, and three NO-modified
Cys, i.e. Cys-S-NO. Based on ESI-MS spectra of
SNO-DDAH-1 recorded after up to 5 days, the relatively unstable
Cys-S-NO presumably formed various new oxidation products
like Cys-S-O2H,
Cys-S-O3H, and others (data not shown). However,
compared with the half-life of small S-nitroso thiols (from
minutes to hours), the half-life of SNO-DDAH-1 in the range of 1 or 2 days is rather high, resembling that of other S-nitrosylated
proteins, e.g. SNO-BSA (39).
View larger version (12K):
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Fig. 2.
Characterization of nitrosated bovine DDAH-1
by absorption spectroscopy and ESI-MS. A, absorption
spectrum of SNO-DDAH-1. B-D, deconvoluted ESI-MS of DDAH-1
upon incubation with 0 (B), 5 (C), and 20 mM (D) DEA NONOate. Non-nitrosated DDAH-1
reveals a mass of 31199 Da (arrow). With the raising of DEA
NONOate concentrations, species with mass differences of 29 (a), 58 (b), and 87 Da (c),
respectively, were formed.
View larger version (16K):
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Fig. 3.
Correlation of the degree of
Cys-S-NO formation with the enzymatic activity of
apo-DDAH-1. DDAH-1 activity was determined in 250 mM
imidazole/HCl, pH 7.4, upon pre-incubation with various concentrations
of DEA NONOate ( ). The number of Cys-S-NOs was determined
by fluorometric S-NO quantification (
).
gt11 phage library of total bovine brain was not
successful,2 to localize the
nitrosated Cys residues the total amino acid sequence of DDAH-1 was
determined on the protein level. The sequence determination was
performed by Edman degradation and/or MS/MS analysis of peptides from
cleavage with trypsin and endoproteinase Glu-C separated on reversed
phase HPLC, and their molecular masses were determined by ESI-MS. The
herein reported amino acid sequence of bovine brain DDAH-1 (Fig.
4) has been deposited into the Swiss-Prot Protein Data Bank and is available under the accession number P56965.
Considering that the N terminus of DDAH-1 is acetylated (19), the
calculated molecular mass of 31157 Da for the apo-protein agrees well
with that determined previously by ESI-MS (18). Thus, similar to DDAH-1
isolated from rat kidney (40) and human liver (41), no further
permanent post-translational modification is present in the bovine
brain protein. Fig. 4 shows the alignment of the bovine DDAH-1 sequence
with the mammalian sequences known to date. The sequence of DDAH from
P. aeruginosa, for which the crystal structure has recently
been determined, is also included (31). The sequence identity of the
mammalian species is 91%; the overall identity of all species present
in Fig. 4 amounts to 28%. In the structure of the procaryotic DDAH,
the residues of the catalytic triad have been identified as Cys-249,
His-162, and Glu-114 (31). By analogy, the corresponding residues in the mammalian species are Cys-273, His-172, and Asp-126 (Fig. 4).
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Fig. 4.
Alignment of available mammalian DDAH-1 amino
acid sequences and the sequence of P. aeruginosa
DDAH. The alignment is based on sequential homology and
secondary structure prediction in comparison with the secondary
structure elements of the crystal structure of P. aeruginosa
DDAH. The sequence of bovine DDAH-1 was obtained by Edman sequencing
with the exception of the underlined residues, which were
obtained by MS/MS. All Cys residues are designated as a
white C in a black box. In
the presented secondary structure analysis, arrows denote
-strands, gray bars
-helices, and
open bars 310-helices. Active site
residues are labeled with an asterisk. NO refers
to nitrosated Cys.
Identification of tryptic SNO peptides of bovine SNO-DDAH-1 by LC-MS
-modules arranged in a 5-fold pseudo-symmetry. A similar
fold was also found for L-arginine:glycine amidinotransferase and L-arginine:inosamine-phosphate
amidinotransferase (31, 45). The corresponding modules in the model
structure of the mammalian DDAH-1 are shown in Fig. 5B.
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Fig. 5.
Model structure of bovine apo-DDAH-1.
A, superposition of the model of bovine DDAH-1
(white) with the crystal structure of P. aeruginosa DDAH (31) (blue). L-Cit is
located in the active site pocket of P. aeruginosa DDAH
(represented as a ball-and-stick model). The N-terminal loop
(residues 14-25) of the procaryotic protein, which closes the active
site upon substrate or product binding, is indicated in
light blue. The corresponding helix-turn-helix
motif (residues 5-29) of the eucaryotic is indicated in
yellow. B, bottom view of the structural model of
DDAH-1. The structure consists of five more or less degenerated
modules (I-V) colored in purple,
blue, green, yellow, and
orange, respectively. The modules are arranged in a 5-fold
pseudo-symmetry. C and D, environments of
C273 (C) and C221 (D) in
the model structure of DDAH-1. In both instances the Cys residues are
surrounded by charged residues. For details see "Discussion."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-modules also in DDAH-1. The
large loop between residues 14 and 25 in module I of P. aeruginosa DDAH (yellow) closes over the active
site when a substrate or a product is bound (31). In contrast, module I
of the mammalian DDAH-1 consists of a
-fold, resulting in a
small helix-turn-helix motif (light blue). This
structural feature is more similar to module I of
L-arginine:glycine amidinotransferase (45), used as a
template for the solution of the P. aeruginosa DDAH structure (31). These differences would represent major structural
differences between the structure of mammalian and the prokaryotic
DDAH.
of
Cys-273 is surrounded by the carboxylic groups of Glu-77 and Asp-78 at
the distance of 3.4 and 5.0 Å, respectively. Moreover, N
or N
atoms of the basic residue His-172
are located as close as 5.0 Å from the S
of Cys-273
(Fig. 5C). A similar situation was found in the case of
Cys-221, which is surrounded by the carboxylic groups of Asp-169 and
Glu-191 at a distance of 5.1 and 6.0 Å, respectively. His-172, located
at the distance of 6.2 Å, seems also to influence the S
of Cys-221 (Fig. 5D). These structural features compare well with the spatial arrangement of the catalytic Cys present in the structural model of methionine adenosyl transferase (46) and in the
crystal structure of caspase-3 (protein data base number 1CP3). Taken
together, it would appear that the spatial proximity of charged
residues in DDAH-1 might be responsible for the activation of Cys-221
and Cys-273 in the nitrosation reaction. However, it should be
emphasized that this interpretation is based on a structural model and
that more studies are needed to support this conclusion.
1 mM of DEA NONOate
(Fig. 1A). Although this donor concentration appears to be a
rather high, because of a short half-life of NO in solution (minutes)
(48) and its slow release with time, the actual concentration of free
NO in solution is much lower. In cellular systems intracellular NO
concentrations can be relatively high. For instance, in neurons the
concentration ranges between 2-4 µM (49), and in
activated neutrophils and macrophages it is below 10 µM
(50). Therefore, it can be assumed that the free NO
concentrations used in this study are of physiological relevance.
of Cys-221 and Cys-273 is of the order of ~9 Å.
However, in the tetrahedral complex, the distance between both
S
atoms should be substantially lower (~3.7 Å).
Consequently, the mutual approach of both residues would require
changes in the protein structure. Indeed, based on the circular
dichroism studies, a structural alteration upon Zn(II) binding
to apo-DDAH-1 takes place (19).
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ACKNOWLEDGEMENT |
---|
We thank Dr. Peter Hunziker from the Protein Analysis Unit, University of Zürich, for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Olga Mayenfisch-Stiftung (to M. K.) and by Swiss National Science Foundation Grant 31-58858.99.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.
The amino acid sequence reported in this paper has been submitted to the Swiss Protein Database under Swiss-Prot accession no. P56965.
To whom correspondence may be addressed. Tel.: 41-1-635-5552; Fax:
41-1-635-6805; E-mail: mknipp@bioc.unizh.ch or
mvasak{at}bioc.unizh.ch.
§ Present address: Functional Genomics Center Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.
¶ Present address: Novartis Research Foundation, Friedrich Miescher Institute, Maulbeerstr. 66, CH-4068 Basel, Switzerland.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M209088200
2 M. Knipp and S. Kozlov, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
NMDA, N-methyl-D-aspartate;
ADMA, L-N,N
-dimethylarginine
(asymmetric dimethylated
L-Arg);
Cit, citrulline;
DDAH, L-N
,N
-dimethylarginine
dimethylaminohydrolase (dimethylargininase);
DEA NONOate, 2-(N,N-dimethylamino)-diazenolate-2-oxide;
DDT, 1,4-dithio-D,L-threitol;
ESI-MS, electrospray
ionization mass spectrometry;
EXAFS, extended x-ray fine structure;
HPLC, high pressure liquid chromatography;
LC-MS, liquid
chromatography/mass spectrometry;
MES, 2-morpholino-ethanesulfonic
acid;
MMA, L-N
-methylarginine
(monomethylated L-Arg);
NOS, nitric oxide synthase;
TCEP·HCl, Tris-(2-carboxyethyl)-phosphine
hydrochloride;
TEA, triethanolamine;
TFA, trifluoroacetic acid.
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