Role of an Intrasubunit Disulfide in the Association State of
the Cytosolic Homo-oligomer Methionine Adenosyltransferase*
Gabino F.
Sánchez-Pérez
,
María
Gasset§,
Juan J.
Calvete¶, and
María A.
Pajares
From the
Instituto de Investigaciones
Biomédicas "Alberto Sols," Consejo Superior de
Investigaciones Científicas (CSIC)-Universidad Autónoma
de Madrid, Arturo Duperier 4, 28029 Madrid, Spain, the
§ Instituto de Química-Física
"Rocasolano," CSIC, Serrano 119, 28006 Madrid, Spain, and the
¶ Instituto de Biomedicina de Valencia, CSIC, Jaime Roig 11, 46006 Valencia, Spain
Received for publication, October 4, 2002, and in revised form, December 12, 2002
 |
ABSTRACT |
Recombinant rat liver methionine
adenosyltransferase has been refolded into fully active tetramers (MAT
I) and dimers (MAT III), using as a source chaotrope-solubilized
aggregates resulting from specific washes of inclusion bodies. The
conditions of refolding, dialysis in the presence of 10 mM dithiothreitol or 10 mM GSH with 1 mM GSSG, allowed the production of both isoforms, the
nature of the redox agent determining the capacity of the final product (MAT I/III) to interconvert. Refolding in the presence of 10 mM dithiothreitol yielded mainly MAT III in a
concentration-dependent equilibrium with the homotetramer
MAT I. However, refolding in the presence of the redox pair GSH/GSSG
resulted in a stable MAT I and III mixture. Blockage of dimer-tetramer
interconversion has been found related to the production of a single
intramolecular disulfide in methionine adenosyltransferase during the
GSH/GSSG folding process. The residues involved in this disulfide have been identified by mass spectrometry and using a set of single cysteine
mutants as cysteines 35 and 61. In addition, a kinetic intermediate in
the MAT I dissociation to MAT III has been detected. The physiological
importance of these results is discussed in light of the structural and
regulatory data available.
 |
INTRODUCTION |
Cysteines are one of the least abundant but highly reactive amino
acids present in polypeptide chains. Their appearance is, in many
cases, related to a key role of the sulfhydryl group on the structural
and functional features of the protein. These side chains have the
capacity to actively participate in catalysis, can be
posttranslationally modified, or can lead to the apparition of redox
sensitive sites that could have effects either on modulation of
catalysis or because of the generation of conformational restrictions (1-3). Posttranslational modifications, such as prenylation and acylation, take place in the sulfhydryl group of cysteine residues located at or near the C terminus of the protein, leading to
translocation from the cytosol to membranes (4, 5). Nitrosylation
occurs in residues related to activity control, either activating
(ryanodine receptor) or inactivating key proteins
(MAT)1 (6). Zinc chelation
determines the acquisition of polypeptide geometries compatible with
the recognition of nucleic acids (zinc fingers) (7). Moreover, the
presence of cysteine sulfhydryl groups may also play an essential role
in folding and association, because of disulfide bond formation (8, 9).
To form this covalent bond, the residues must come to a distance such
that the C
atoms of the cysteines are within 3.8-4.5 Å of each
other (9). These disulfides can be established within the same subunit, linking different areas of the polypeptide chain that may be quite distant in the sequence, or between subunits, binding each monomer on
the right position to the others (10, 11).
The production and stability of disulfides have been related to the
existence of an oxidative environment. Thus, the number of
extracellular or secreted proteins that contain disulfide bridges in
their structure is large, because of their passage through a highly
oxidative environment, such as that of the endoplasmic reticulum, where
the presence of thiol/disulfide oxidoreductases favors their production
(12). However, in vitro experiments have demonstrated that
it is possible to obtain disulfide bonds even under strong reducing
conditions, provided the local concentration of sulfhydryls is high
enough (13). Therefore, the presence of disulfides in cytosolic
proteins is possible, and in fact, there have been some reports
describing their existence (14, 15). The role for these covalent bonds
is not clear and may be related to the control of protein activity.
One of the few cases where the presence of a cytosolic disulfide has
been described is the liver-specific methionine adenosyltransferase (MAT), a homo-oligomeric protein that is isolated as stable tetramers (MAT I) and dimers (MAT III), differing in the affinity for methionine (16). MAT I is 10-fold more active than MAT III under physiological concentrations of the amino acid (60 µM) (17), and hence
the ratio of isoforms determine the activity level displayed in the cell. This is of special relevance if we take into account that the
product of MAT reaction is S-adenosylmethionine, the main methyl donor for the transmethylation reactions (18). This enzyme presents 10 cysteine residues/subunit (19, 20), and under several
in vitro and in vivo conditions it has been
possible to demonstrate their role on the activity and oligomeric
state: (a) inactivation and dissociation of MAT I is
produced by N-ethylmaleimide (NEM) modification of two
SH
groups (21, 22); (b) site-directed mutagenesis of
Cys69 renders the enzyme mainly as dimers (23);
(c) all of the mutants on the cysteines comprised between
residues 35 and 105 have some effect on the MAT I/MAT III ratio (23);
(d) the enzyme activity is inhibited by GSSG (2) and
nitrosylation of Cys121 (24, 25); (e) inhibition
of the glutathione synthesis leading to a 30% reduction in the GSH
levels, and hence to the alteration of the GSH/GSSG ratio, correlates
with a decrease in MAT activity (26); and (f) low MAT
activity and mainly dimers are detectable under the oxidative
conditions of alcohol liver cirrhosis (27). Moreover, a disulfide bond
between Cys35 and Cys61 that are located in the
-sheet of contact between dimers, according to the crystal
structure, has been identified (15, 28). This disulfide was not
detected when the protein was overexpressed in Escherichia
coli, where MAT I/III appears in a
concentration-dependent equilibrium (29), thus suggesting a
role for it on the association. The aim of this paper is to demonstrate
the key role of the disulfide on MAT assembly; this, to the best of our
knowledge, is the first description of such a role for this type of
covalent bond in a cytosolic oligomer.
 |
EXPERIMENTAL PROCEDURES |
Materials
Methionine, ATP, phenylmethanesulfonyl fluoride, pepstatin A,
aprotinin, leupeptin, antipain, DTT, ampicillin, GSH, GSSG, cyanogen
bromide, 4-vinylpyridine, iodoacetamide, and the molecular mass
standards for gel filtration chromatography were products from
Sigma. NEM and trypsin sequencing grade were purchased from Serva (Heidelberg, Germany) and Roche Diagnostics (Barcelona, Spain), respectively. [2-3H]ATP (20 Ci/mmol) and [1,4
14C]NEM (2-10 mCi/mmol) were supplied by Amersham
Biosciences and ARC Inc. (St. Louis, MO), respectively.
Isopropyl-
-D-galactopyranoside was a product of Ambion
(Austin, TX). Phenyl Sepharose CL-4B and Superose 12 HR 10/30 were
purchased from Amersham Biosciences. Optiphase HiSafe 3 scintillation
fluid was obtained from E & G Wallac (Milton Keynes, UK). Cation
exchanger AG-50W-X4, goat anti-rabbit IgG-horseradish peroxidase,
Bio-Rad protein assay kit I, and the electrophoresis reagents were from
Bio-Rad. YM-30 ultrafiltration membranes and glass microfiber filters
GF/B were purchased from Amicon Corp. (Beverly, MA) and Whatman Ltd.
(Maidstone, UK), respectively. Chemiluminescence Renaissance reagents
and BCA were obtained from PerkinElmer Life Sciences and Pierce,
respectively. Urea, Me2SO, and Triton X-100 were purchased
from Merck. The rest of the buffers and reagents were of the best
quality commercially available.
Methods
Rat Liver MAT Expression, Refolding, and
Purification--
Competent E. coli BL21(DE3) cells were
transformed with the plasmid pSSRL-T7N, which contains the sequence of
wild type MAT, or the mutants for the cysteine residues of the protein
(23). The cells were grown on LB medium plus ampicillin and expression induced by the addition of
isopropyl-
-D-galactopyranoside (30). The inclusion
bodies were isolated after disruption of the cells by sonication and
purified as described by López-Vara et al. (31).
Refolding was carried out using either 10 mM DTT or a redox
buffer composed by GSH/GSSG (10:1) at 10 mM GSH.
Purification of the DTT-refolded proteins was carried out as previously
described but using a Q-Sepharose column (31), whereas
GSH/GSSG-refolded MATs needed an additional purification step on phenyl
Sepharose. The purity of the samples was tested by SDS-PAGE, and the
final protein samples were characterized by fluorescence and circular dichroism spectroscopies.
MAT Association/Dissociation Studies by Analytical Gel
Filtration and Phenyl Sepharose Chromatographies--
Purified samples
were concentrated by ultrafiltration through YM-30 membranes to obtain
the desired range of protein concentrations. Samples of 100 µl were
injected on a Superose 12 HR 10/30 gel filtration column connected to
an Advanced Protein Purification System (Waters) and eluted using 50 mM Tris/HCl, pH 8, 10 mM MgSO4 (buffer A) containing 150 mM KCl at a flow rate of 0.3 ml/min. A280 was recorded, and 210-µl
fractions were collected for their use in dot blot and MAT activity
measurements. The peak and shoulder corresponding to GSH/GSSG-refolded
MAT III and I, respectively, were concentrated and reloaded on the same
column, and their elution volumes were determined as above.
In parallel, 2-ml samples of the same protein concentrations were
loaded on 3-ml phenyl Sepharose columns equilibrated in buffer A,
washed with 20 ml of the equilibration buffer, and eluted with 10 ml of
buffer A containing 50% (v/v) Me2SO. Only the first 5-ml
fractions of sample loading (MAT I) and Me2SO elution (MAT III) were used for dot blot, MAT activity, and protein concentration measurements. Moreover, to follow dissociation MAT I fractions were
collected, diluted 5-fold with buffer A, and incubated for 30 min prior
to reloading on new phenyl Sepharose columns. Again fractions
corresponding to MAT I and MAT III were collected and used for dot
blot, MAT activity, and protein concentration measurements.
Dissociation Followed by ANS Binding--
A 50 mM stock solution of ANS in methanol was prepared, and its
concentration was determined using
370 = 6800 M
1 cm
1 (32). ANS was added to a
20-µl MAT I sample (1-4 mg/ml) to render a final concentration, upon
dilution (1:50), of 40 µM, and to pre-equilibrated MAT
III solutions (1 ml) of 20-80 µg/ml. Controls for each case were
prepared accordingly, and the reaction was carried out for 1 h at
room temperature in the dark. The final methanol concentration in the
samples was 0.08% (v/v). Fluorescence emission at 470 nm was recorded
for 8 min upon excitation at 380 nm in a photon counting SLM-8000
spectrofluorometer. MAT I kinetics were recorded immediately after
dilution with a dead time 5 s. The data were corrected for base
line and instrumental factors and adjusted to one- or two-phase
exponential decays using the program GraphPad Prism (GraphPad Software
Inc., San Diego, CA) and the following equations.
|
(Eq. 1)
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|
(Eq. 2)
|
where Y is the fluorescence intensity at 470 nm;
A is the amplitude of the signal; t is the time;
k, X, and Z, the rate constants; and
Y
is the fluorescence intensity at an
infinite time.
Large-zone Gel Filtration Chromatography--
The dissociation
constants were determined using a Biogel A gel filtration column
(0.7 × 9 cm) equilibrated in buffer A containing 150 mM KCl. The column was run at 25 °C at a flow rate of
0.23 ml/min, and the accuracy of the flow was controlled by weighting fractions collected before and after each individual run (33). Purified
proteins, wild type and mutants, were concentrated by ultrafiltration,
as described above, to obtain the desired range of protein
concentrations (0.05-5 mg/ml), and 3.5 ml of large zones were
collected. Loading was performed at least 1 h after sample
preparation to ensure equilibrium. Elution was followed by
A280 and dot blot analysis of the 75-µl
fractions collected. The data obtained by both methods were used to
determine the centroid volume for each zone using GraphPad Prism. The
centroid volumes were then used to calculate the weight-average
partition coefficient (
w) and the corresponding dimer
fraction (fD) according to the following equations.
|
(Eq. 3)
|
|
(Eq. 4)
|
where Vc is the centroid volume for the large
zone; Vo is the void volume; Vi
is the included volume of the column;
T is the partition
coefficient for the tetramer; and
D is the partition
coefficient of the dimer determined experimentally.
A plot of fD versus log of the dimer
concentration allows calculation of the equilibrium constant.
fD values were also used to determine the free
energy of the process using equations analogous to those described by
Park and Bedouelle (34) for a dimer-monomer equilibrium.
|
(Eq. 5)
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|
(Eq. 6)
|
The total free energy of the process, from MAT I to the unfolded
protein, can be calculated using the following expression.
|
(Eq. 7)
|
where fD is the fraction of protein as dimer;
c is the protein concentration; R is the gas
constant; T is the absolute temperature;
G is
the free energy of the process;
G1 is the free energy of the tetramer-dimer conversion;
G2 is the free energy calculated for the
dimer-monomer unfolding; m in this particular case is the
slope of the dependence of the thermodynamical parameter upon protein
concentration;
Gtot is the free energy of the
total process, tetramer to monomer unfolding; and K is the
equilibrium constant.
Dot Blot--
The samples of the column fractions
(maximum volume, 30 µl) were spotted on nitrocellulose membranes.
After denaturation using 6 M guanidinium chloride (50 µl), the membrane was washed twice with TTBS (20 mM Tris/HCl, pH 7.5, 500 mM NaCl, 0.05% (v/v)
Tween 20), before the blocking step using low fat dry milk (3% w/v). The membrane was washed again with TTBS and incubated with a 1:20000 (v/v) solution of an anti-MAT polyclonal antibody prepared in our
laboratory using DTT-refolded MAT. Under these conditions the only band
detected corresponds to MAT in SDS-PAGE gels. The membranes were
revealed using Renaissance, the exposed films were subjected to
densitometric scanning, and the data were used for the corresponding calculations.
Determination of the Free Sulfhydryl Content and Location of the
Disulfide Bond--
The number of free
SH groups for wild type and
mutant MATs was determined as described previously using NEM labeling
(15, 22). In addition, for quantitation of free cysteine residues and
disulfide bonds in wild type and mutant MAT proteins, samples of the
purified proteins were dialyzed against ammonium acetate extensively
and lyophilized, and the content of reduced and oxidized cysteine
residues was determined by mass spectrometry. To this end, ~1 mg/ml
of protein in 150 mM Tris/HCl, pH 8.6, 1 mM
EDTA, 6 M guanidinium chloride was heated for 5 min at
80 °C, cooled down to room temperature, incubated either with 10 mM iodoacetamide for 1 h at room temperature or with
1% 2-mercaptoethanol for 2 min at 100 °C, followed by the addition
of a 5-fold molar excess of 4-vinylpyridine or iodoacetamide over
reducing agent, and incubated for 1 h at room temperature. The
samples were dialyzed against deionized MilliQ water and lyophilized.
Aliquots of these samples were subjected to matrix-assisted laser
desorption ionization time-of-flight (MALDI-TOF) mass spectrometry
using a PE Biosciences Voyager DE-Pro instrument and sinapinic acid
(saturated in 0.1% trifluoroacetic acid in 50% acetonitrile) as matrix.
To locate free cysteines and disulfide bonds within the primary
structure of MAT (Swiss-Prot S06114), native and ethylpyridylated proteins (10 mg/ml in 70% formic acid) were degraded with cyanogen bromide (100 mg/ml) overnight in the dark under a nitrogen atmosphere. In addition, native and carboxyamidomethylated proteins (1 mg/ml in 100 mM ammonium bicarbonate, pH 8.3, 10 mM
iodoacetamide) were degraded with sequencing grade trypsin at an
enzyme/substrate ratio of 1:50 (w/w) overnight at 37 °C. Mass
fingerprinting of the digests was done by MALDI-TOF mass spectrometry
using
-cyanocinnamic acid (saturated in 0.1% trifluoroacetic acid
in 50% acetonitrile) as matrix.
N-terminal Sequencing--
N-terminal sequence analyses of the
purified wild type and mutant MAT proteins were done using an Applied
Biosystems 473A sequencer following the manufacturer's instructions.
The results indicated that 70% of the protein had a complete
N-terminal sequence (MNGPVDGL), whereas the rest had lost the initial
methionine. These data were taken into account in the MALDI-TOF mass
spectrometry calculations.
Sedimentation Velocity Experiments--
The samples (0.1-5
mg/ml) were loaded on the An50Ti rotor of a Beckman Optima XL-A
analytical ultracentrifuge (Beckman Instruments Inc.) equipped with
absorbance optics, and the experiments were performed at 50,000 rpm and
18 °C. Absorbance scans (step size, 0.005 cm) were taken at 280 nm.
The sedimentation velocity data were analyzed with the program Svedberg
(35), and the sedimentation velocity coefficients were corrected for
solvent composition and temperature to obtain
s20,W (36).
Determination of the Protein Concentration--
Protein
concentration of the samples after ultrafiltration was measured using
the Bio-Rad kit I and using bovine serum albumin as a standard.
However, protein concentration on the fractions collected from
analytical phenyl Sepharose columns was determined after
trichloroacetic acid precipitation using the BCA system, because
of the presence of Me2SO.
MAT Activity Measurements--
MAT activity was measured as
described by Gil et al. (37) using 160-µl samples of the
column fractions. Kinetics for methionine and ATP were performed in the
concentration range comprised from 1 µM to 10 mM for one of the substrates while keeping the other constant at 5 mM (2). Oxidation constants and GSSG
inhibition were measured as previously described (2, 38).
 |
RESULTS |
Association/Dissociation Processes in Liver-specific
MAT--
Rat liver MAT has been shown to appear in a
concentration-dependent equilibrium upon its overexpression
in E. coli cytosol. However, there are no indications that
such equilibrium takes place in the rat liver-purified enzyme forms. In
an attempt to clarify the difference among these proteins and get
insight into the structure-function facts that regulate MAT
oligomerization, we have used two refolding systems described
previously in our laboratory. The use of DTT rendered only dimers at
the protein concentrations used for refolding, but concentration by
ultrafiltration caused the apparition of tetramers (Fig.
1, A and B). On the
other hand, replacement of DTT by a GSH/GSSG redox buffer allowed the production of a mixture of tetramers and dimers, which do not associate
upon enhancement of the protein concentration (Fig. 1, C and
D). Reloading of both GSH/GSSG-refolded MAT I and III on gel
filtration chromatography showed no variation in the elution position
for both association states (Fig. 1E). These results were
confirmed on analytical phenyl Sepharose chromatography, a procedure
that allows the quantitative separation of MAT I and III forms by means
of differences in hydrophobicity (39). Association was also studied by
sedimentation velocity, but the use of this technique was precluded
because complete dissociation of tetramers to dimers and monomers was
already observed at the highest protein concentrations tested (2 mg/ml).

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Fig. 1.
Gel filtration chromatography analysis of
wild type DTT- and GSH/GSSG-refolded samples. Wild type MAT was
refolded using the DTT or GSH/GSSG refolding methods. Samples of the
refolded mixtures (100 µl) were injected on a Superose 12 HR column
at two protein concentrations and detected by measuring MAT activity
( ) and by densitometric scanning of the dot blots corresponding to
each collected fraction ( ). A and B show the
profiles for DTT-refolded MAT at 0.2 mg/ml (A) and 2 mg/ml
(B); C and D depict the profiles for
the GSH/GSSG-refolded MAT at 0.2 mg/ml (C) and 2 mg/ml
(D). E shows the profile for the rechromatography
of MAT I ( and ) and III ( and ) from D (the
data have been scaled for graphical purposes). Elution positions for
rat liver purified MAT I (point a) and III (point
b) are indicated in A. The figure shows the results of
a typical experiment.
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Dissociation of MAT I was then studied by analytical phenyl Sepharose
chromatography. As can be observed, isolated DTT-refolded MAT I render
tetramers and dimers upon reloading on the hydrophobic columns, whereas
GSH/GSSG-refolded MAT I appears as a stable tetramer (Fig.
2, A and F). More
information about the dissociation process was then obtained by
fluorescence spectroscopy of ANS-bound DTT-refolded MAT I samples (Fig.
3A). Dilution followed by
immediate recording of the fluorescence emission at 470 nm revealed the
presence of an exponential decay, until the emission levels
corresponding to an ANS-bound dimer were reached (Fig. 3B).
This decay was preceded by a short lag phase, suggesting the presence
of at least one intermediate in the process (Fig. 3A,
inset). The fluorescence intensity of MAT I corresponded to
the double of that for MAT III, thus indicating that the dye binds in
an area not related to association. This change cannot be followed by
intrinsic fluorescence, because no significant changes in the Trp and
Tyr emissions were detectable (data not shown). The same is true for
other spectroscopic techniques, such as circular dichroism, because MAT
I and III show identical spectra. The data were then analyzed using
either one- or two-phase exponential decays (Equations 1 and 2), with the best fit being obtained by a single exponential. The calculated rate constant (koff) for dissociation to MAT III
is 0.022 s
1, and the calculated half-life for MAT I upon
dilution is 14.69 ± 0.5 s at 25 °C. Large-zone gel
filtration chromatography was then used for the calculation of the
dissociation constants (Fig. 4). Using
large zones in the 0.05-5 mg/ml range, the centroid volumes for each
protein concentration were calculated and used to determine the
weight-average partition coefficients (Equation 3) and the dimer
fraction (Equation 4). These data were then used to obtain the
Kd value for the DTT-refolded MAT, which was found
to be in the 105 M
1 range (Table
I). Moreover, the free energy of
association was calculated using this dissociation constant in Equation 6, as well as using the dimer fractions in Equations 5 and 6 (Table I).

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Fig. 2.
Analytical phenyl Sepharose chromatography of
DTT- and GSH/GSSH-refolded tetrameric forms of MAT wild type and
mutants. Wild type MAT and cysteine mutants were refolded using
the DTT and GSH/GSSG refolding systems and concentrated to 1 mg/ml.
Tetramer forms for each sample were separated by phenyl Sepharose
chromatography and diluted in the equilibration buffer before reloading
on analytical phenyl Sepharose columns. The presence of tetramer and
dimer forms was followed by measuring MAT activity. Fractions
1-3 correspond to sample loading and the washing step, whereas
fractions 4 and 5 are the elution with
Me2SO. A-E show the behavior of DTT-refolded
tetrameric forms of MAT wild type and mutants, whereas F-J
depict the results of the GSH/GSSG-refolded tetramers. A and
F correspond to the wild type; B and G
correspond to the C35S mutant; C and H correspond
to the C61S mutant; D and I correspond to the
C57S mutant; and E and J correspond to the C69S
mutant. The figure shows the results of a typical experiment.
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Fig. 3.
Kinetics of MAT I dissociation followed by
ANS fluorescence. Wild type MAT was DTT-refolded and purified. A
concentrated sample (4 mg/ml), MAT I, was incubated with ANS and
diluted to 0.08 mg/ml immediately before recording fluorescence at 470 nm (A). The inset shows an enlargement of the
initial phases of the reaction. Samples of MAT III (0.08 mg/ml) were
also incubated with ANS, and their fluorescence was recorded
(B). The data were fitted to one- or two-phase exponential
decays as described under "Experimental Procedures."
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Fig. 4.
Determination of the
Kd for wild type MAT by large- zone gel
filtration chromatography. Large zones for several protein
concentrations (0.05-5 mg/ml) were loaded on a gel filtration column.
A280 was recorded, and fractions of the eluent
were collected for its use in dot blot. The centroid volume for each
zone was determined and used for the calculation of the weight-average
partition coefficient and the fD. The
Kd value was calculated from the plot of
fD versus log of the dimer concentration
(µM). The figure shows a plot of a typical experiment for
DTT-refolded wild type MAT.
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Table I
Dissociation constants and free energies of the association process
The Kd values were calculated using the fraction of
dimer deduced from the centroid volume of the large-zone gel filtration
chromatography, using a protein concentration range from 0.05 to 5 mg/ml. G(H2O) was calculated by two methods.
G(H2O)1 was obtained using
Kd values in Equation 6, whereas
G(H2O)2 was calculated from the same
dimer fractions using equations analogous to those of Park and
Bedouelle for a monomer-dimer equilibrium.
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Role of the Sulfhydryl Groups in MAT Association--
MAT contains
10 cysteine residues/subunit that could be present either in a reduced
or partially oxidized pattern. In the present study different
oxido/reduction conditions were used for refolding of wild type MAT,
and thus several parameters related to the redox status of the protein
could differ among the forms obtained. Analysis of the free
SH group
content of the different MAT forms was carried out by NEM or
vinylpyridine labeling followed by mass spectrometry (Table
II). The molecular mass of
DTT-refolded MAT was 43,518 Da, which changed to 44,549 Da after
treatment with vinylpyridine under denaturing but nonreducing
conditions (Fig. 5, A and
B). This value was not altered by reduction of the protein
followed by ethylpyridilation. The mass difference of 1031 Da
corresponded to the addition of 10 (1031/106 = 9.7) EP groups,
thus indicating that the 10
SH groups of the DTT-refolded MAT subunit
were accessible. On the other hand, the molecular mass of the
GSH/GSSG-refolded MAT I and III increased by 823 Da upon treatment with
vinylpyridine under denaturing but nonreducing conditions, and by 1061 Da when the proteins were fully reduced and ethylpyridilated (Fig.
5C). These results showed that the GSH/GSSG-refolded
proteins contained eight titrable cysteine residues and a disulfide
bond.
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Table II
Molecular and enzymatic features of wild-type refolded-MAT
I/III
Wild type MAT samples were refolded with DTT or GSH/GSSG, and the
oligomeric forms obtained were characterized and analyzed for their
SH content and their behavior against GSSG and GSH/GSSG buffers. The
data on the table are the means ± S.D. of the results obtained in
experiments carried out in triplicate for the refolded MAT I/III.
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Fig. 5.
MALDI-TOF mass spectrometry. Mass
spectrometric determination of the molecular masses of DTT-refolded MAT
before (A) and after (B) treatment with
vinylpyridine under denaturing but nonreducing conditions is shown.
Molecular masses of GSH/GSSG-refolded MAT I and III (C) and
of C35S and C61S MAT mutants (D) upon treatment with
vinylpyridine under denaturing but nonreducing conditions are also
included.
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The redox behavior of these proteins was further characterized. Wild
type MAT refolded using both procedures is inhibited by GSSG, but the
Ki values calculated are lower for the GSH/GSSG-refolded forms, indicating a higher susceptibility to this
agent (Table II). In addition, modulation of the activity by redox
buffers is also observed, and the Kox values
were calculated on the basis of the previous knowledge for this enzyme
(Table III). These values are close to
the R[GSH] data determined in vivo under mild to
severe oxidative stress (300-3 mM) (40).
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Table III
Characterization of the refolded MAT III forms
Wild type MAT and mutants C35S and C61S were refolded in the presence
of either DTT- or GSH/GSSG-containing buffers. The dimeric forms were
purified in each case and characterized kinetically. In addition,
samples were used for free SH determination by MALDI-TOF. The data
shown are the means ± S.D. of three independent experiments
carried out in triplicate.
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C35S and C61S Mutations Abrogate the Oligomer Interconversion
Blockage--
The results presented above prompted us to explore the
possibility that the presence of a disulfide bond could be responsible for the differences observed among DTT- and GSH/GSSG-refolded MATs. For
this purpose, individual mutants on cysteine residues located at the
dimer-dimer contact area, according to the crystal structure of the
protein, were selected. Of the 10 cysteine residues of the MAT
polypeptide chain, four are located at the
-sheet (B2) of contact
between dimers (Cys35, Cys57,
Cys61, and Cys69) (Fig.
6). Among those, Cys61 and
Cys69 are specific for the liver enzyme and hence may be
directly linked to the special behavior shown by this MAT. These four
residues were mutated to serine, and the recombinant proteins were
refolded using either the DTT or GSH/GSSG system described above and
purified. Characterization by CD and fluorescence spectroscopy showed
no difference as compared with the wild type protein. However, whereas the specific activity recovered upon refolding paralleled that for each
mutant in the cytosolic fraction, purification of the refolded proteins
led to changes in the enzymatic behavior; those related to C69S are
especially important. This mutant shows low activity (20% of the wild
type) in the cytosolic fraction and appears mainly as dimers, whereas
once DTT-refolded and purified its specific activity increases to about
64% (70 nmol/min/mg) of that shown by the wild type, MAT and tetramers
can be detected. Moreover, all of the refolded mutants have sigmoidal
kinetics, but the affinity shown by C61S for both substrates was
reduced (Table III).

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Fig. 6.
Localization of Cys35,
Cys57, Cys61, and Cys69 in rat
liver MAT I crystal structure. The figure shows the two strands of
-sheet B2, where Cys57, Cys61, and
Cys69 are located (Protein Data Bank code 1QM4). In
addition, the position of Cys35 on -helix 1 is also
included. Sulfhydryl groups are shown in green, and the
dashed lines indicate the hydrogen bonds stabilizing the
-sheet. In gray are the positions that Cys57
and Cys69 should have to form a disulfide
bond.
|
|
The capacity of association of the DTT- and GSH/GSSG-refolded mutants
was then explored. As can be observed, all of them are in
tetramer-dimer equilibrium upon DTT refolding, a behavior that is
conserved in C35S and C61S after GSH/GSSG refolding (Fig. 2). The
molecular masses of these MAT mutants, determined after vinylpyridine labeling by MALDI-TOF mass spectrometry were 44,455 Da (Fig.
5D), indicating the presence of nine free
SH groups in
C35S and C61S under both refolding conditions (Table III). Dissociation
constants were also calculated by large-zone gel filtration
chromatography, the values being larger than that for the wild type MAT
(Table I). On the other hand, stable MAT I/III forms were obtained when using the redox buffer system to refold C57S and C69S (Fig. 2). The
fact that the association process is still observed in the absence of
Cys35 and Cys61 is consistent with a role for
these residues in the oligomerization process.
Identification of the Disulfide Bond in
GSH/GSSG-refolded MAT--
To ensure that a disulfide
bridge between Cys35 and Cys61, such as that
detected in liver-purified MAT, is responsible for the blockage of
association, free sulfhydryls of GSH/GSSG-refolded wild type MAT were
ethylpyridylated (EP) or carboxyamidomethylated (CM), and the native
and the EP- and CM-GSH/GSSG-refolded wild type MAT proteins were
degraded with CNBr and trypsin. The proteins and their digestion
products were analyzed by MALDI-TOF mass spectrometry. The
isotope-averaged (±8 Da) molecular masses of native and
EP-GSH/GSSG-refolded wild type MATs were 43,507 and 44,328 Da,
respectively (Table IV). The mass
difference of 824 Da indicated the presence of 7.8 titrable sulfhydryl
groups. On the other hand, both reduced and EP-GSH/GSSG-refolded wild
type MAT proteins showed the same molecular mass of 44,565 Da (Table
IV). Thus, fully reduced MAT proteins contained 10 EP-cysteine
residues. As a whole, these results indicated the presence of one
disulfide bond and eight sulfhydryl groups in GSH/GSSG-refolded wild
type MAT. CNBr degradation of GSH/GSSG-refolded MAT yielded a fragment
of 5816 Da, which corresponded to the stretch 21-75 with
Met65 as sulfoxide. This polypeptide contains four cysteine
residues, those at positions 35, 57, 61, and 69. On the other hand, the fragment 21-77 with Met65 sulfoxide and two
EP-cysteines (M + H+ 6230) was characterized upon CNBr
degradation of EP-GSH/GSSG-refolded MAT. Hence, GSH/GSSG-refolded MAT
contained two free cysteines and a disulfide bond within the stretch
comprising residues 21-75. When carboxyamidomethylated
GSH/GSSG-refolded MAT was digested with trypsin and analyzed by mass
fingerprinting MALDI-TOF mass spectrometry, tryptic fragments
containing CM-cysteine residues 69, 105, 121, 150, 312, and 377 were
found (Table IV). On the other hand, the ion of
m/z = 6588.3 corresponded to the polypeptide stretch 2-62 with two CM-cysteines, one disulfide bond, and
Met20 oxidized. These results indicate the existence
of a disulfide bond involving two cysteines among 9, 35, 57, and 61. Furthermore, the ion of m/z = 2551.9 was
assigned to fragments 34-48 disulfide bonded to 55-62, containing a
CM-cysteine and a disulfide bond. This allowed the identification of
Cys35 as one of the residues forming part of the disulfide
with either Cys57 or Cys61. These results,
together with the fact that Cys61, but not
Cys57, is at S-S bonding distance from Cys35
in the crystal structure (Protein Data Bank accession code 1QM4) (Fig.
6), strongly support the possibility that Cys35 and
Cys61 are disulfide-bonded in GSH/GSSG-refolded MAT.
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|
Table IV
Results of the tryptic digestion analyzed by MALDI-TOF mass
spectrometry
Fragments assigned by MALDI-TOF mass spectrometry in GSH/GSSG-refolded
MAT III digested with trypsin under denaturing but nonreducing
conditions in the presence of 10 mM iodoacetamide.
|
|
 |
DISCUSSION |
MAT isoforms, as purified from the liver, appear as stable dimers
and tetramers, whereas the recombinant protein, as isolated from
E. coli, shows a concentration-dependent
equilibrium between MAT I and III (29). These behaviors are paralleled
by the MAT forms generated under two different refolding systems, DTT-
and GSH/GSSG-based (31), thus providing a useful tool to explore the
mechanisms that control association. Analysis of the association process by analytical ultracentrifugation techniques evidenced a
dramatic sensitivity of the oligomeric assemblies to centrifugal forces. This phenomenon, experimentally manifested as dissociation, has
been described in other oligomeric proteins, such as
NAD-dependent dehydrogenases and tryptophan synthase (41).
This instability can be explained by the low number of interactions
between dimers shown in the MAT I crystal structure (28) (Fig.
7). In fact, only five polar interactions
have been described to take place between dimers in contrast to what
happens in the E. coli MAT (c-MAT) (42). In this last case
the number of interactions between dimers in the tetramer structure is
far larger than in MAT I, and no dimer structures have been obtained to
date. Moreover, the particular arrangement of interactions observed is
completely different to that shown in the crystal structure of c-MAT
(Fig. 7). Rat liver presents a squared contact area formed by residues of its four subunits that is in direct contact with the solvent. On the
other hand, c-MAT has this squared area surrounded by a larger
contact area that precludes its exposure to the solvent, thus protecting those interactions and allowing a higher stability for the complex. Based on these considerations the techniques of
choice for our experiments were gel filtration and analytical phenyl Sepharose chromatographies.

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Fig. 7.
Dimer-dimer contact areas in c-MAT and rat
liver MAT I. The figure shows two views of the dimer-dimer contact
areas for E. coli (Protein Data Bank code 1FUG) and rat
liver MATs (Protein Data Bank code 1QM4), the only two proteins of this
family whose crystal structure has been determined. Subunits are shown
under different colors: magenta (A),
red (B), green (C), and
blue (D).
|
|
Hydrophobic chromatography was described by Kunz et al. (39)
as a way to separate MAT I and III, because of the high avidity shown
by MAT III for phenyl Sepharose, and has been used as a quantitative
method to establish the ratio of isoforms (29). Therefore, association
and dissociation of the refolded MATs were followed by this method,
whereas calculation of the dissociation constants was performed using
large-zone gel filtration chromatography as described by Beckett (33).
The Kd values obtained either for wild type and
mutants were in the 105 M
1 range,
a value comprised between those described for proteins showing
association/dissociation behavior (43). These values are in agreement
with those previously obtained for the cytosolically overexpressed wild
type MAT (29) and much lower than that for c-MAT, which was calculated
to be 1010 M
1 (44). In addition,
Kd values were also used to calculate the free
energy of association to MAT I, and the values in all of the cases were
of the same order,
7 to
8 kcal/mol, again similar to the values
obtained for proteins showing association processes (33, 45). These
refolded forms present a higher susceptibility to inhibition by GSSG as
compared with the liver purified-MAT, but their oxidation constants are
in the range for a regulation under mild to severe oxidative stress
(40). On the other hand, the calculated half-life for the tetramer is
shorter than that previously published for the cytosolically
overexpressed MAT (14.69 versus 858 s), where the
temperature is the main difference between both experiments (29).
Differences among the refolding systems used are based in
their redox potential; thus it could be possible that the opposite association behavior shown by DTT- and GSH/GSSG-refolded MAT may be due
to an effect related to the redox state of the cysteine sulfhydryls.
MAT polypeptide contains 10 such residues/subunit that under the redox
buffer could originate reduced or partially oxidized proteins (19, 20).
Our results indicate that two of the 10
SH groups are not accessible
to alkylation, even after guanidinium chloride denaturation, thus
suggesting their involvement in a disulfide. These residues have been
identified using single cysteine mutants as Cys35 and
Cys61, the same residues involved in the disulfide bond
identified in rat liver-purified MAT isoforms (15). The conclusions
from these studies with mutant proteins are in line with mass
spectrometry analysis of CNBr and trypsin degradation products of EP-
or CM-GSH/GSSG-refolded MAT. Moreover, GSH/GSSG refolding of these
mutants preserves the concentration-dependent equilibrium,
clearly indicating the importance of Cys35 and
Cys61 in the association. The presence of these cysteines
in the
-sheet of contact between dimers and the fact that
Cys61 is a liver-specific amino acid may be related to the
special features shown by the liver-specific MAT, such as its capacity to generate two oligomeric assemblies (16). In fact, the sulfhydryls of
these two cysteines appear perfectly oriented and at bonding distance
in the crystal structure of MAT I (obtained in the presence of DTT)
(28). As for Cys57 and Cys69 (another
liver-specific cysteine), both are also located in the
-sheet B2 and
close enough as to form a disulfide bond, but their sulfhydryl groups
are facing opposite directions (Fig. 6). Thus, it could be possible
that under certain circumstances this second disulfide is formed, but
for this purpose a slight torsion of the main chain of the protein
would be needed. Therefore, it can be deduced that alterations in this
-sheet of contact could be responsible for the changes observed in
the oligomerization.
Intramolecular disulfides are known to have a role in protein
oligomerization (46) but are not commonly observed in such a reducing
environment as cellular cytosol (10, 14). However, during folding it is
possible that a large increase in the local sulfhydryl concentration
occurs, allowing the production of disulfides. Moreover, in
vitro experiments have shown the production of optimal oxidation
rates during folding, even in the presence of significant concentrations of reductants (13). In vivo, using alcohol
liver cirrhotic samples, an increase in the oxidation conditions is also observed that was caused by a 30% reduction in the GSH levels. Under these conditions a decrease in MAT activity correlated with its
apparition mainly as dimers has been described (27), but no explanation
for this incapacity to associate has been obtained for the moment.
Based on our data, we suggest that under cirrhotic oxidative conditions
the production of dimers may be favored by allowing the disulfide bond
(Cys35-Cys61) to form, before association to
MAT I takes place. Another possibility that cannot yet be excluded is
the production of another disulfide (Cys57-Cys69) or of both disulfide bridges
(Cys35-Cys61 and
Cys57-Cys69) in cirrhotic MAT dimers. In these
cases, association may not be precluded exclusively by the presence of
the disulfide bonds but also by the incapacity of establishing the
correct polar interactions on the interface, because of the main chain
torsion. Moreover, a third possibility exists: the presence of two
types of dimers. Our results on the kinetics of MAT I dissociation
suggest the presence of an intermediate in the pathway to MAT III. Even
when it is not possible to infer the oligomeric state of such an
intermediate, it is attractive to suggest that it may be a dimer just
separated from MAT I, before undergoing the small changes that lead to
MAT III. These changes should occur close to the ANS-binding site, located between Pro358-Gly359 and the sequence
Val-Gly-Ala that limits the active-site loop as suggested by
Sánchez del Pino et al. (47), because a kinetic intermediate is detectable by this modification. All of these data
together showing the effects exerted by dissociation on areas related
to the active site, such as the flexible loop and the central domain
may explain the 10-fold decrease in methionine affinity shown by MAT
III as compared with MAT I or its activation by Me2SO (2,
17).
Using the previous knowledge on MAT III urea unfolding (47, 48) and the
results described herein, we propose the following model for MAT I
folding and association.
The unfolded MAT polypeptide (U) associates to the
dimer (MAT III) through a monomeric equilibrium-intermediate
(I), which evolves to a monomeric kinetic intermediate
(I
). The dimer (MAT
III) could then undergo oxidation of Cys35 and
Cys61 to make a disulfide bond that precludes further
association or evolve to a kinetic intermediate
(I
) that leads to the tetramer
(MAT I). The production of the Cys35-Cys61
disulfide in MAT I must then take place in this last kinetic intermediate before definitive association in the tetramer. The total
free energy of the process can be calculated using Equation 7, and in
our case this renders a
G(H2O) of 24.41 kcal/mol.
Finally, all of these results indicate that the presence of a disulfide
bond between Cys35 and Cys61 is responsible for
the stabilization of tetramer and dimer forms, blocking the association
process. The production of this disulfide takes place at the
dimer level (MAT III and I
).
 |
ACKNOWLEDGEMENTS |
We thank Francisco Garrido for technical
assistance, Dr. J. L. Neira for the critical comments of the
manuscript, and Dr. D. Laurents and B. Ashley Morris for grammatical
and English style corrections.
 |
FOOTNOTES |
*
This work was supported by Fondo de Investigación
Sanitaria Grant 01/1077 (to M. A. P.), Dirección
General de Investigación Científica y Técnica Grant
PM 97/0064 (to M. A. P.), Ministerio de Ciencia y Tecnologia
Grant BMC 2002-00243 (to M. A. P.), and Neuropharma Grants SA-CSIC
(to M. G. and M. A. P.) and BMC2001-3337 (to J. J. C.).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.:
34-91-5854414; Fax: 34-91-5854401; E-mail: mapajares@iib.uam.es.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M210177200
 |
ABBREVIATIONS |
The abbreviations used are:
MAT, ATP:L-methionine adenosyltransferase;
DTT, dithiothreitol;
NEM, N-ethylmaleimide;
ANS, 8-anilinonapththalene-1-sulfonic
acid;
EP, ethylpyridylated;
c-MAT, E. coli methionine
adenosyltransferase;
MALDI-TOF, matrix-assisted laser desorption
ionization time-of-flight;
CM, carboxyamidomethylated.
 |
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