From the Division of Hepatology and Gene Therapy, University of
Navarra, Irunlarrea 1, 31008 Pamplona, Spain, the ¶ University of
Tennesse Medical School, Veterans Affairs Medical Center/Research 151, Memphis, Tennessee 38104, and the Laboratory of Molecular
Biology, National Institute of Mental Health,
Bethesda, Maryland 20892-4034
Received for publication, October 3, 2000, and in revised form, January 26, 2001
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ABSTRACT |
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Methionine adenosyltransferase (MAT) catalyzes
the synthesis of S-adenosylmethionine (AdoMet), the main
alkylating agent in living cells. Additionally, in the liver, MAT is
also responsible for up to 50% of methionine catabolism. Humans with
mutations in the gene MAT1A, the gene that encodes the
catalytic subunit of MAT I and III, have decreased MAT activity in
liver, which results in a persistent hypermethioninemia without
homocystinuria. The hypermethioninemic phenotype associated with these
mutations is inherited as an autosomal recessive trait. The only
exception is the dominant mild hypermethioninemia associated with a G-A transition at nucleotide 791 of exon VII. This change yields a MAT1A-encoded subunit in which arginine 264 is replaced by
histidine. Our results indicate that in the homologous rat enzyme,
replacement of the equivalent arginine 265 by histidine (R265H) results
in a monomeric MAT with only 0.37% of the AdoMet synthetic activity. However the tripolyphosphatase activity is similar to that found in the
wild type (WT) MAT and is inhibited by PPi. Our in
vivo studies demonstrate that the R265H MAT I/III mutant
associates with the WT subunit resulting in a dimeric R265H-WT MAT
unable to synthesize AdoMet. Tripolyphosphatase activity is maintained in the hybrid MAT, but is not stimulated by methionine and ATP, indicating a deficient binding of the substrates. Our data indicate that the active site for tripolyphosphatase activity is functionally active in the monomeric R265H MAT I/III mutant. Moreover, our results
provide a molecular mechanism that might explain the dominant inheritance of the hypermethioninemia associated with the R264H mutation of human MAT I/III.
Methionine adenosyltransferase
(MAT,1 EC 2.5.1.6.) is a key
metabolic enzyme that catalyzes the synthesis of the most important biological alkylating agent, S-adenosylmethionine (AdoMet)
(1). The synthesis of AdoMet occurs in a two-step reaction. In the first step AdoMet and PPPi are synthesized from the
substrates methionine and ATP. Subsequently the tripolyphosphate
generated is hydrolyzed to PPi and Pi before
the products of the reaction are released (2, 3). The function of the
tripolyphosphatase activity in the overall reaction catalyzed by MAT is
still under discussion (4). In mammalian tissues three forms of MAT
have been described that are the products of two distinct genes (5-8). The gene MAT2A encodes a 396-amino acid catalytic subunit
expressed in extrahepatic tissues, as well as in fetal liver and
hepatocarcinoma, that associates with a regulatory Two decades or more ago, it was observed that several newborn children,
screened for hypermethioninemia as an indicator of homocystinuria due
to a deficiency in cystathionine Materials--
Columns and chromatography media were from
Amersham Pharmacia Biotech. AdoMet was from Boehringer Ingelheim
(Knoll). All other reagents were from Sigma.
Site-directed Mutagenesis--
A 1.2-kilobase fragment
containing the rat MAT1A coding region (33) was subcloned
into a pET vector. The resulting plasmid includes a 5'-sequence that
encodes for 6 histidine residues and a thrombin cleavage site in frame
with the rat liver MAT1A coding region. Mutants were
obtained by inverse polymerase chain reaction according to the
procedure of Pérez-Mato et al. (34). The mutants were
identified by sequencing the complete MAT cDNA.
Expression and Purification of His-tagged WT and MAT I/III
Mutants--
WT and MAT I/III mutants were overexpressed in E. coli BL21(DE3) as described previously (33). Recombinant
His-tagged WT and MAT I/III mutant proteins were purified from the
bacterial cytosolic extracts by affinity chromatography on a
Ni2+-Sepharose column equilibrated in 50 mM
Tris/HCl, pH 8, 0.5 M NaCl, 75 mM imidazole.
Elution was performed using a linear gradient from 75 to 500 mM imidazole in the same buffer. His-tagged MAT I/III
proteins elute from the Ni2+ column at 250 mM
imidazole. Protein purity was always more than 95% as estimated by
SDS-PAGE (35). Imidazole was removed by dialysis against 10 mM Tris/HCl, pH 7.5, 150 mM KCl, 5 mM DTT. No changes in the fluorescence, CD spectra,
oligomeric state, and enzymatic activity were observed after digestion
of the recombinant proteins with thrombin (data not shown). Therefore
all the assays were carried out using the His-tagged recombinant proteins.
Association between WT and R265H MAT I/III Mutant
Subunits--
To study the formation of heterodimers in
vivo, bacteria were cotransformed with the plasmid pSSRL (12) to
produce the WT MAT subunit and a pET vector with the coding region of
the R265H MAT mutant to produce a His-tagged protein. His-tagged
proteins were purified from the cytosolic extracts of cotransformed
bacteria following the procedure indicated above. The molecular
mass of the purified protein was estimated by size exclusion
chromatography. To assess the subunit composition, the recombinant
protein was denatured by incubation with 5 M urea in 50 mM Tris/HCl, pH 7.5, 150 mM KCl, 10 mM MgCl2, 5 mM DTT. Then, the
mixture was chromatographed on a 1-ml Ni2+-Sepharose column
equilibrated with 50 mM Tris/HCl, pH 8, 0.5 M
NaCl, 75 mM imidazole. After loading the sample, the column was washed with 10 column volumes of the same buffer. Elution was
carried out with 5 column volumes of 50 mM Tris/HCl, pH 8, 0.5 M NaCl, 0.5 M imidazole, and 0.5-ml
fractions were collected. Protein elution was monitored by measuring
the absorbance at 280 nm. The protein-containing fractions were pooled,
and aliquots were analyzed by size exclusion chromatography and
SDS-PAGE (35). Heterodimer formation was also tested in
vitro in two ways. In the first, MAT III purified from rat liver
was incubated with equimolar concentration of the R265H mutant
His-tagged protein (0.2 mg/ml) in 50 mM Tris/HCl, pH 7.5, 150 mM KCl, 10 mM MgCl2, 5 mM DTT for 30 min at 25 °C. Alternatively, MAT III
purified from rat liver was denatured by incubation with 5 M urea in 50 mM Tris/HCl, pH 7.5, 150 mM KCl, 10 mM MgCl2, 5 mM DTT, and then refolded by 20-fold dilution (the final
concentration of WT MAT subunits was 0.6 µM) in the same
buffer containing a 3-fold molar excess of His-tagged R265H MAT I/III
mutant subunit, followed by incubation for 30 min at 25 °C. The two
mixtures were then re-purified on a Ni2+ column as
indicated above. Imidazole was removed from the retained fractions
using a 5-ml Hi-trap desalting cartridge. Protein was then denatured by
adding urea to a final concentration of 5 M and
chromatographed again on a 1-ml Ni2+-Sepharose column
equilibrated with 50 mM Tris/HCl, pH 8, 0.5 M
NaCl, 75 mM imidazole. Flow-through and imidazole eluted
fractions were analyzed, in both experiments, by SDS-PAGE (35).
Purification of MAT from Rat Liver--
MAT III was purified
from rat liver according to the procedure described previously (36).
Protein purity was more than 95% as estimated by SDS-PAGE (35).
Size Exclusion Chromatography--
Protein samples were analyzed
using a Superdex 200 HR 10/30 column equilibrated with 50 mM Tris/HCl, pH 7.5, 150 mM KCl, 10 mM MgCl2 in an AKTA fast protein liquid
chromatography (Amersham Pharmacia Biotech). After sample
injection, proteins were isocratically eluted at a flow rate of 0.8 ml/min. Fractions of 0.2 ml were collected. Protein elution was
monitored by measuring the absorbance at 280 nm. The elution volume (in
ml) of the standard proteins were: tiroglobulin (669 kDa), 8.1;
ferritin (440 kDa), 10.22; catalase (232 kDa), 12.11; ovalbumin (43 kDa), 14.73; chymotripsin A (25 kDa), 18; ribonuclease A (13.7 kDa),
19. According to the elution volume of the standard proteins, the
estimated molecular mass of the R265H MAT I/III rat mutant
protein was 41 kDa. AdoMet synthetic as well as tripolyphosphatase
activities were measured in the collected fractions.
Enzymatic Activity Measurements--
Activity assays were
performed in a final volume of 100 µl, at 37 °C for 10 min in 50 mM Tris/HCl, pH 8, 4 mM MgCl2, 250 mM KCl, 3.6 mM DTT. 2 mM
ATP/MgCl2 and methionine or 2 mM
tripolyphosphate were used as substrates to determine AdoMet synthetase
or tripolyphosphatase activities, respectively. Both activities were
monitored by following the formation of inorganic phosphate according
to the method of Lanzetta (37). The effect of NO on the
tripolyphosphatase activity of the R265H mutant was determined in the
absence of DTT. Activation of the tripolyphosphatase activity by
substrates was studied by enzyme preincubation with 2 mM
methionine and 2 mM ATP.
Oligomeric State of the R265H MAT I/III Mutant Protein--
The
molecular mass and catalytic properties of a purified R265H MAT
I/III mutant protein have been studied by size exclusion chromatography. The elution volume of the R265H MAT was 16 ml (Fig.
1), which corresponds to a molecular
mass of 41 kDa according to the elution profile of standard
proteins. This result suggests that this mutant protein is a monomeric
MAT.
Kinetics of the Monomeric R265H MAT I/III Mutant--
To test the
enzymatic activity of the R265H MAT I/III mutant protein, fractions
were collected, and AdoMet synthetic activity and tripolyphosphatase
activity were measured. Interestingly, the analysis of the R265H
fractions revealed that, although no AdoMet synthetic activity was
detected, a tripolyphosphatase activity peak was measured at the same
elution volume as the absorbance peak (Fig. 1). This result indicates
that tripolyphosphate hydrolysis can be catalyzed by a single MAT
subunit. In contrast, AdoMet synthetic activity requires at least a
dimeric enzyme, since this active site is constituted between two monomers.
The catalytic properties of the R265H MAT mutant were then further
studied. MAT and tripolyphosphatase activities of the MAT mutant R265H
were measured at 2 mM of methionine and ATP or
tripolyphosphate (Fig. 2). Both
activities were determined as the accumulation of inorganic phosphate
(Pi) after incubation at 37 °C for different periods of
time. Our data indicate that while the Vmax for
the tripolyphosphatase activity of the R265H mutant was very similar to
that determined for a WT enzyme, AdoMet synthetic activity of this MAT
variant was decreased by more than 99% (Table
I). Replacement of arginine 265 by serine
instead of histidine resulted in a monomeric MAT protein (data not
shown) with more than 99% reduction of the AdoMet synthetic activity
and a 5-fold decrease in the tripolyphosphatase activity (Table I),
suggesting that the positive charge of the residue at position 265 is
involved in the hydrolysis of PPPi. Tripolyphosphatase
activity of the R265H MAT I/III mutant was dependent on
Mg2+ concentration, but K+ was not required
(not shown). No change of tripolyphosphatase specific activity was
observed by increasing the protein concentration (0.026-0.345 mg/ml).
Additionally, to verify that the monomer does not reassociate under
assay conditions, a gel filtration molecular mass determination
in a column equilibrated with 2 mM PPPi was
performed. Under these conditions the estimated molecular mass
of the R265H MAT mutant was 41.6 kDa (not shown). These two lines of evidence indicate that the monomeric state of this MAT mutant is maintained after incubation with PPPi, and
therefore, the tripolyphosphatase activity of the R265H MAT mutant is
not a consequence of subunit dimerization. In contrast to WT protein, preincubation with methionine and ATP did not stimulate the
tripolyphosphatase activity (not shown). R265H tripolyphosphatase
activity is specific for PPPi. Less than 2% of the
hydrolytic activity measured in the presence of PPPi was observed when
ATP, PPi, or metatripolyphosphate were used as substrates
(Table II). No significant changes of enzymatic activity or Km were detected when the
tripolyphosphatase activity of the R265H MAT I/III mutant was tested in
the presence of ATP (Fig. 3). However, a
decrease of the enzymatic activity from 128 to 67 nmol
min Association between WT and R265H Mutant MAT I/III
Subunits--
The capacity of the R265H MAT I/III mutant to form
hetero-oligomers with a WT subunit has been examined both in
vivo and in vitro, taking advantage of the N-terminal
His-tag of the mutant protein. If such an interaction occurred, mutant
and WT subunits would combine in a hetero-oligomeric form that would be
retained on a Ni2+-Sepharose column. When MAT purified from
bacteria overexpressing WT and R265H MAT subunit is chromatographed on
a Ni2+-Sepharose column, all the protein binds to the
column, as shown by SDS-PAGE analysis of the flow-through and the 0.5 M imidazole eluted fraction (Fig.
4A). However, when the enzyme
is unfolded with urea before being reanalyzed by Ni2+
chromatography, the protein re-distributes between the flow-through and
retained fractions (Fig. 4B). Therefore, the original
protein must be a hetero-oligomer constituted by WT and His-tagged
mutant subunits.
Hetero-oligomeric formation was also demonstrated in vitro.
When MAT III was incubated with equimolar concentrations of R265H mutant, the Ni2+ column did not retain any MAT III subunit,
suggesting that there was no association between the mutant and the rat
liver protein after 30-min incubation (Fig.
5A). Alternatively,
hetero-oligomer formation was attempted under conditions more nearly
approaching those that might exist in vivo by refolding a
urea-denatured MAT III in the presence of the R265H mutant. After
purification and urea denaturation of the resulting His-tagged protein,
the flow-through and Ni2+-retained fraction of a second
Ni2+-Sepharose chromatography were analyzed by SDS-PAGE.
Protein was detected in both fractions (Fig. 5B), indicating
that the His-tagged purified protein was a hetero-oligomer consisting
of refolded MAT III and mutant (His-tagged) subunits.
Hybrid MAT Characterization--
To estimate the molecular
mass of the hetero-oligomers resulting from cotransformed
bacteria, size exclusion chromatography was performed, 0.4-ml fractions
were collected, and AdoMet synthetic and tripolyphosphatase enzymatic
activities were measured. The estimated molecular mass of MAT III
purified from rat liver, which was used as a control, was 91 kDa,
according to the dimeric state of this protein. Peaks of AdoMet
synthetic and tripolyphosphatase activities were detected when
fractions of MAT III were analyzed (Fig.
6A). The molecular mass
of the purified hetero-oligomeric MAT was of 90 kDa, indicating its
dimeric nature. Similarly to MAT III, the protein-containing fractions
had tripolyphosphatase activity, but, in contrast, no AdoMet synthetic
activity was detected (Fig. 6B). Therefore, MAT purified by
Ni2+ chromatography from cotransfected bacteria is a
heterodimer with an impaired AdoMet synthetic capacity, although its
tripolyphosphatase activity is maintained. The absence of MAT activity
in the heterodimeric WT R265H MAT might explain the dominant
inheritance of the phenotype associated with the R264H mutation
described elsewhere (26). To further assess the subunit composition of
the heterodimeric MAT, the flow-through and retained fractions
resulting from a Ni2+-Sepharose chromatography of a
denatured heterodimer were analyzed by size exclusion chromatography.
The imidazole-eluted protein had a molecular mass of 41 kDa and
maintains the tripolyphosphatase activity, but no synthesis of AdoMet
was detected (Fig. 7A). The molecular mass, inability to oligomerize, and the absence of
AdoMet synthetic activity indicate that this enzyme is the His-tagged R265H MAT mutant. However, the protein obtained in the flow-through had
a molecular mass of 90 kDa, and both the tripolyphosphatase and
AdoMet synthetic activities were recovered (Fig. 7B). These findings confirm that this protein is a non-His-tagged WT MAT subunit
that refolds and oligomerizes to get the native, fully active
conformation. Additionally, refolding of WT MAT subunits in the
presence of R265H MAT mutant results in the formation of WT R265H
heterodimers (not shown). It has been shown previously that the
tripolyphosphatase activity of MAT III is activated by preincubation of
the enzyme with the substrates methionine and ATP (36). To test whether
activation occurs also upon preincubation of the WT R265H heterodimeric
MAT, the protein was pretreated with 2 mM methionine and 2 mM ATP, and tripolyphosphatase activity was then measured.
No activation was observed after incubation of the heterodimer with
methionine and ATP. The activity values were 132 and 134 nmol
Pi min Methionine adenosyltransferase catalyzes the synthesis of AdoMet,
the main alkylating agent in living cells (1). In the liver, MAT is
also responsible for the catabolism of up to 50% of the dietary
methionine (15). Humans with mutations in the gene MAT1A
have decreased MAT activities in the liver, resulting in persistent
hypermethioninemia without homocystinuria (22, 23). The
hypermethioninemic phenotype associated with all mutations tested to
date is inherited as an autosomal recessive trait, the only known
exception being the hypermethioninemia due to the G-A transition at
nucleotide 791 of exon VII (21-30). This change results in a mutant
MAT1A-encoded protein in which arginine 264 has been replaced by histidine (26). To investigate the biochemical basis of the
dominant inheritance of hypermethioninemia of individuals with this
mutation, we have studied the enzyme kinetics and the oligomerization
capacity of a purified MAT protein containing the homologous R265H
mutation encoded by rat MAT1A.
Our results indicate that the molecular mass of the R265H MAT
I/III rat mutant protein is 41 kDa, establishing its monomeric state.
Although previous studies using cell extracts also suggested that the
equivalent R264H MAT I/III mutant protein cannot form homo-oligomers
(26, 39), it has been reported that the tetrameric conformation of the
E. coli MAT is not altered by the replacement of the
equivalent arginine 244 by histidine (4). Our results demonstrate that
the R265H MAT I/III rat mutant has impaired ability to form
homo-oligomers. Arginine 265, similarly to arginine 244 in the E. coli MAT, is involved in a salt bridge formation with symmetrical
glutamic 58 (31, 32), which is important for dimerization (40).
Therefore, the absence of this arginine might compromise the
dimerization capacity of this MAT mutant.
The synthetic reaction catalyzed by MAT occurs through two consecutive
steps. AdoMet and PPPi are first synthesized from
methionine and ATP; PPPi is subsequently hydrolyzed to
PPi and Pi to allow product release from the
active site of the enzyme (2, 3). The function of the
tripolyphosphatase activity of MAT is still under discussion (4, 41).
MAT activity of the R265H MAT I/III mutant was less than 1% of the
activity of the WT enzyme, in agreement with previous data, which
indicate that the active site of a dimeric MAT is configured by amino
acid residues from both subunits (31, 32, 40). However, the
tripolyphosphatase activity was not modified by this mutation,
indicating that the active site for this activity is fully functional.
Therefore, the hydrolytic site for the tripolyphosphatase activity must
be configured by residues held on a single subunit. Tripolyphosphatase
activity of this MAT mutant is similar to that determined for a WT
enzyme in the resting, less active state (36). Additionally, we found
that tripolyphosphatase activity of the R265H MAT I/III mutant is not regulated by NO, which further agrees with the mutant enzyme being in
the resting state. We have shown previously that tripolyphosphatase activity of MAT III is stimulated by preincubation with methionine and
ATP (36). However, no activation of the tripolyphosphatase activity was
found when R265H MAT was preincubated with methionine and ATP,
suggesting that the regulation of this activity by the natural
substrates of the enzyme has been lost. The absence of AdoMet synthetic
activity, and the failure of methionine and ATP to activate the
tripolyphosphatase activity, suggest that binding of the substrates is
impaired. Substitution of arginine 265 by serine instead of histidine
resulted in a 5-fold decrease of tripolyphosphatase activity. Thus, it
seems that, although the presence of a positive charge at position 265 is involved, it is not an absolute requirement for the PPPi
hydrolytic activity. This result might be explained by previous
evidence, which indicates the positive charge of the equivalent
arginine 244 in the E. coli MAT is responsible for the
correct orientation of the PPPi in the active site of the enzyme (4, 42).
Tripolyphosphatase activity of the R265H MAT I/III mutant is specific
of PPPi, and depends on the presence of Mg2+ in
the assay mixture, but K+ is not required. These findings
suggest that while Mg2+ is directly involved in the binding
of PPPi (31), K+, which binds to the interface
between monomers (40), might contribute to the stabilization of a
functional conformer of dimeric MAT, which is not accessible to the
monomeric R265H mutant. This mutant showed no cooperativity when its
tripolyphosphatase activity was assayed with different PPPi
concentrations. Tripolyphosphatase activity of WT MAT is not altered by
the presence of saturating concentrations of ATP (36). This finding
might be explained by assuming that ATP and PPPi bind to
different sites, or, alternatively, binding of the PPPi
moiety of ATP might be sterically restricted in the dimeric enzyme. To
further assess this question, we studied the effect of ATP on the
tripolyphosphatase activity of the monomeric R265H MAT mutant. We found
that ATP had no effect on the tripolyphosphatase activity of the R265H
MAT I/III mutant. Since accessibility should not be compromised in the
monomer, we propose that ATP and PPPi have different
binding sites. In contrast, PPi, a classical inhibitor of
MAT tripolyphosphatase activity, induces a 1.5- and 2.3-fold decrease
of the enzymatic activity and affinity of the enzyme, respectively.
Similarly to E. coli MAT (43), PPi and
PPPi might compete for the same binding site. Our data
suggest that the active site of MAT has two coordinated subsites: the
synthetic site, configured by amino acid residues from both subunits
(31, 32), is responsible for the binding of methionine and ATP and
performs the AdoMet synthetic reaction. The hydrolytic site, configured by amino acid residues from one single subunit, accounts for the binding of PPPi and performs the PPPi
hydrolytic reaction.
Dominant inheritance of the hypermethioninemia of humans carrying the
R264H mutation in MAT1A might be explained by our findings, which indicate that replacement in the rat homologue of arginine 265 by
histidine produces a monomeric MAT that interacts with the WT MAT
subunit, in vivo and in vitro, resulting in a
hybrid enzyme with impaired AdoMet synthetic activity. Evidence
suggesting the capacity of the MAT mutant R264H to associate with WT
MAT was first proposed for the human enzyme by Chamberlin et
al. (26) on the basis of MAT activity recovered in COS cell
extracts after cotransfection with two vectors expressing human R264H
mutant and WT MAT, respectively. We have demonstrated that MAT enzyme purified by Ni2+-Sepharose chromatography from E. coli cotransformed with plasmids expressing rat R265H mutant and
WT MAT is a hetero-oligomer, which is formed by mutant and WT subunits.
In contrast, when MAT III purified from rat liver was incubated with
the recombinant rat R265H MAT I/III mutant, no association was observed
under the experimental conditions used in our studies. This apparent
discrepancy might be explained by assuming that the dissociation
constant for the dimer is sufficiently low to prevent MAT III-mutant
subunit exchange under our experimental conditions. Since monomers
generally fold to nearly the final conformation before the association
step (44), association between MAT subunits may occur at a late
intermediate step of the folding process. Indeed, we found that a
hybrid MAT, containing WT and mutant subunits, was formed when a
urea-unfolded MAT III purified from rat liver was refolded in the
presence of the R265H MAT I/III mutant. The presence of urea might
overcome the proposed kinetic restrictions, allowing subunit exchange
either by increasing the dimer Kd or by altering the
protein conformation.
To estimate the oligomeric state of the hetero-oligomeric MAT species,
size exclusion chromatography studies were performed, and fractions
were collected to determine their catalytic capacity. The calculated
molecular mass of MAT III was 91 kDa in agreement with its
dimeric structure (5), and AdoMet synthetic as well as
tripolyphosphatase activities were found when the protein-containing fractions were assayed. The hetero-oligomeric MAT purified from E. coli expressing WT and R265H MAT mutant has a molecular
mass of 90 kDa. Since this MAT was previously retained on a
Ni2+-Sepharose column, and the R265H MAT I/III mutant has
lost the ability to form homo-oligomers, the 90-kDa protein must
correspond to a heterodimer containing one R265H mutant (His-tagged)
and one WT subunit. The heterodimer might be stabilized by the salt bridge interaction between arginine 265 in the WT subunit and glutamic
58 in the mutant subunit. The heterodimeric MAT maintains the
tripolyphosphatase activity, but no synthesis of AdoMet was determined
after incubation with methionine and ATP. This lack of AdoMet synthetic
activity might be a consequence of a non-native association between
monomers, resulting in an altered active site and, consequently, in a
deficient substrate binding or catalytic capacity of the heterodimer.
This hypothesis is further supported by the finding that, in contrast
to WT enzyme (36), tripolyphosphatase activity of the R265H-WT MAT is
not activated when the enzyme is preincubated with methionine and ATP.
The ability of the monomeric R265H MAT I/III mutant to associate with a
WT subunit, resulting in a hybrid WT R265H MAT without AdoMet synthetic
activity, might explain the dominant inheritance of the
hypermethioninemia associated with the human R264H MAT I/III mutation.
Additionally, denaturation of heterodimeric MAT by incubation with 5 M urea results in two forms of the enzyme, which were
separated by Ni2+ chromatography, and showed different
molecular mass and enzymatic activities upon refolding. The
retained fraction was a monomer (41 kDa) with tripolyphosphatase
activity, and the flow-through was a dimer (90 kDa) with both
tripolyphosphatase and AdoMet synthetic activity as determined by size
exclusion chromatography. According to their properties, these two
forms of MAT must be identified as refolded His-tagged, monomeric R265H
mutant and dimeric, fully active WT MAT. These findings indicate that
urea unfolding of MAT is a reversible process and further support that
MAT purified from E. coli expressing WT and R265H MAT mutant
is a heterodimer constituted by WT and mutant subunits.
In conclusion, our data provide a molecular explanation for the
dominant inheritance of the persistent hypermethioninemia associated
with the human R264H MAT I/III mutation. We demonstrate that the
equivalent R265H mutation in rat MAT I/III results in a monomeric MAT,
which can associate with the WT enzyme to form a dimeric R265H-WT MAT
lacking AdoMet synthetic activity. We have also shown that the active
site for the tripolyphosphatase activity is functionally active in the
monomeric R265H MAT I/III mutant. Our data suggest that the active site
of MAT has two coordinated subsites: the synthetic site, configured by
amino acid residues from both subunits, that performs the AdoMet
synthetic reaction, and the hydrolytic site, configured by amino acid
residues from one single subunit, that performs the PPPi
hydrolytic reaction.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
subunit to form
MAT II (6, 9). The gene MAT1A encodes a 395-amino acid
subunit, expressed in adult liver, that organizes into dimers, MAT III,
and tetramers, MAT I (5, 10-14). The reason for the presence of these
two different isoenzymes in liver has not been yet elucidated, although
it may be an adaptation to the metabolic requirements of the liver. The liver has the highest specific activity of MAT, which agrees with the
observation that up to 85% of all methylation reactions and as much as
50% of methionine catabolism occur in this tissue (15). Based upon the
different kinetic properties of MAT I and MAT III isoforms, MAT III has
been considered the liver-specific enzyme. While MAT I, similarly to
MAT II in extrahepatic tissues, may maintain the basal AdoMet levels
required by cells, MAT III would be responsible for the clearance of
methionine after a load of this amino acid.
-synthase activity, presented a
persistent hypermethioninemia with normal plasma levels of homocystine
and tyrosine and without severe liver disease (16-19). Enzymatic
studies demonstrated that the origin of this isolated persistent
hypermethioninemia was a severe depletion of MAT activity in liver,
whereas the activity of MAT II in erythrocytes, lymphocytes, and
fibroblasts of these individuals was normal (16, 17, 19, 20). Clinical
and metabolic features in more than 30 such patients have been
described (20-23). The characterization of the human MAT1A
gene led to the detection of 17 different mutations that cause the
previously reported depletion of MAT activity in liver (24-28),
designated, therefore, as MAT I/III deficiency. Segregation and
mutation analysis revealed that in most of the individuals with
MAT1A mutations the hypermethioninemic phenotype is
transmitted as an autosomal recessive trait (22-25, 28). However, a
dominant inherited form of this abnormality has been reported in five
families (21, 22, 26, 28-30). In each such family a G-A transition at
nucleotide 791 was detected in one MAT1A allele. This change
results in a MAT in which arginine 264 is replaced by histidine (26).
Crystallographic studies of Escherichia coli and recombinant
rat liver MAT show that the equivalent arginine 244 or 265, respectively, is located in the interface between the two subunits of
the dimeric enzyme and is involved in a salt bridge with glutamic 42, or the homologous 58 in the rat enzyme, of the symmetric subunit, which
contributes to the stabilization of the oligomeric state of MAT (31,
32, 40). Moreover, arginine 244 contributes to each active site and is
located in the immediate vicinity of the polyphosphate group of ADP (4,
31). It has been shown that replacement of arginine 264 by histidine in
human MAT inactivates the enzyme. Moreover, it has been proposed that this mutation hinders normal oligomeric formation (26). However, substitution of arginine 244 by leucine or histidine in E. coli MAT resulted in an inactive enzyme, which, in contrast,
remains tetrameric with no apparent changes in the secondary structure (4). To understand the biochemical basis of the dominant inheritance of
the phenotype associated with the R264H mutation of human MAT I/III, we
have purified and characterized the homologous rat R265H MAT I/III
mutant. Our data indicate that the active site for the tripolyphosphatase activity is functionally active in the monomeric R265H MAT I/III mutant and provide a molecular mechanism that might
explain the dominant inheritance of the hypermethioninemia associated
with the R264H mutation in human MAT I/III.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Analysis by size exclusion chromatography of
the R265H MAT I/III mutant. The molecular mass of the
His-tagged R265H MAT I/III mutant, that had been purified by
Ni2+-Sepharose column chromatogaphy, was estimated by size
exclusion chromatography on a Superdex 200 HR 10/30 column. ,
absorbance profile at 280 nm. Fractions of 0.2 ml were collected and
tripolyphosphatase (open circles) and AdoMet (closed
circles) activities were measured as described under
"Experimental Procedures." The estimated molecular mass
of the R265H MAT mutant is 41 kDa.
1 mg
1 was
observed when the tripolyphosphatase activity was measured in the
presence of 2 mM PPi, a classical inhibitor of
the tripolyphosphatase activity of MAT (Fig. 3). Since the Hill
coefficient was always close to 1.0, the experimental data were fitted
to the Michaelis-Menten equation. The Km for
PPPi was increased from 84 to 143 µM when the
tripolyphosphatase activity was measured in the presence of
PPi. NO regulates hepatic MAT activity through specific
interaction with cysteine residue 121 (33, 38). To analyze the effect of NO on the tripolyphosphatase activity of the R265H MAT I/III mutant,
the enzyme was nitrosylated by incubation with 250 µM nitrosylated glutathione before determining its capacity to hydrolyze tripolyphosphate. No variations of the activity were observed after
nitrosylation of this enzyme. The enzymatic activities were 125 and 129 nmol Pi min
1
mg
1for the non-nitrosylated and nitrosylated
forms, respectively.
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Fig. 2.
AdoMet synthetic and tripolyphosphatase
activity of the R265H MAT I/III mutant. Tripolyphosphatase
(open circles) and MAT (closed circles)
activities of a purified R265H MAT I/III mutant were measured by
following the formation of Pi after incubation with the
substrates for different periods of time. Tripolyphosphatase and MAT
activity values were 129 and 2 nmol min 1
mg
1, respectively.
Enzymatic activity and estimated Km values for PPPi of
mutants and wild type recombinant rat liver MAT
Substrate specificity of the monomeric MAT mutant R265H
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Fig. 3.
Inhibition of the tripolyphosphatase activity
of the R265H MAT I/III mutant by PPi. The inhibition
of tripolyphosphatase activity of the R265H MAT I/III mutant by ATP and
PPi was examined. Tripolyphosphatase activity of the R265H
mutant was measured in the absence (open circles) and in the
presence of 5 mM ATP (closed circles) or 5 mM PPi (triangles). The experimental
data were fitted to the Michaelis-Menten equation. Values of
Vmax and Km for
PPPi were 128 nmol min 1
mg
1 and 83 µM, respectively, in
the absence of any inhibitor; 67 nmol min
1
mg
1 and 143 µM, respectively,
in the presence of PPi; and 129 nmol
min
1 mg
1 and 78 µM, respectively, in the presence of ATP.
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Fig. 4.
In vivo association between WT and
His-tagged R265H MAT mutant subunits. Proteins were obtained and
purified as described under "Experimental Procedures."
Hetero-oligomers were chromatographed on a Ni2+-Sepharose
column before (A) or after (B) denaturation with
5 M urea. The presence of MAT protein in the flow-through
fraction of the affinity column was indicative of association between
WT and His-tagged mutant subunits, which were eluted with 500 mM imidazole. Proteins were identified by SDS-PAGE.
FT, flow-through fraction from the
Ni2+-Sepharose column; R, fraction retained on
the Ni2+-Sepharose column; Mr, molecular
mass standards (bovine serum albumin, 81 kDa; ovalbumin, 47.7 kDa; carbonic anhydrase, 34.6 kDa).
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Fig. 5.
In vitro association between MAT
III purified from rat liver and recombinant His-tagged R265H mutant
subunits. Association between purified MAT III and R265H MAT
mutant subunits was analyzed under native conditions (A) or
by refolding of MAT III in the presence of mutant subunits
(B). The obtained protein mixtures were then denatured with
5 M urea and chromatographed on a
Ni2+-Sepharose column. The presence of MAT in the
flow-through fraction of the affinity column was indicative of
association between MAT III and His-tagged mutant subunits, which were
eluted with 500 mM imidazole. Proteins were identified by
SDS-PAGE. FT, flow-through fraction from the
Ni2+-Sepharose column; R, fraction retained on
the Ni2+-Sepharose column; Mr, molecular
mass standards (standard proteins were the same as in Fig.
4).
1
mg
1 for the non-preincubated and preincubated
forms, respectively. The absence of activation of the heterodimeric MAT
might be explained by a deficient or non-productive interaction with
the substrates. This hypothesis would agree with the previous result,
indicating that the synthesis of AdoMet is impeded in the
heterodimer.
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Fig. 6.
Size exclusion chromatography of MAT III and
hetero-oligomeric MAT. The molecular mass of the
hetero-oligomeric MAT was estimated by size exclusion chromatography of
the protein purified by Ni2+-Sepharose chromatography. ,
absorbance profile at 280 nm. Fractions of 0.4 ml were collected, and
tripolyphosphatase (open circles) and AdoMet synthetic
(closed circles) activities were measured as described under
"Experimental Procedures." A, rat liver MAT III.
B, hetero-oligomeric MAT. The estimated molecular
mass for MAT III and the hetero-oligomer was 90 kDa.
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Fig. 7.
Size exclusion chromatography of
heterodimeric MAT subunits. Heterodimeric MAT purified from
E. coli expressing WT and His-tagged R265H mutant was
denatured in 5 M urea, and the resulting subunits were
separated by chromatography on a Ni2+-Sepharose column. The
flow-through and retained fractions were analyzed by size exclusion
chromatography on a Superdex 200 HR 10/30 column equilibrated without
urea. A, retained fraction; B, flow-trough. ,
absorbance profile at 280 nm. Fractions of 0.4 ml were collected, and
tripolyphosphatase (open circles) and AdoMet synthetic
(closed circles) activities were measured as described under
"Experimental Procedures." The estimated molecular mass of
the retained fraction and flow-through was 41 and 90 kDa,
respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by grants from the Plan Nacional de I+D (SAF 98/132), Departamento de Salud del Gobierno de Navarra (5697), National Institutes of Health Grant AA-12677, and by 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 Spanish Ministerio de Ciencia y
Tecnología.
§ Supported by a Contrato de reincorporación from the Spanish Ministerio de Ciencia y Tecnología.
** To whom correspondence should be addressed: Division of Hepatology and Gene Therapy, University of Navarra, Irunlarrea 1, 31008 Pamplona, Spain. Tel.: 34-948-425678; Fax: 34-948-425677; E-mail: fjcorrales@unav.es.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M009017200
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ABBREVIATIONS |
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The abbreviations used are: MAT, methionine adenosyltransferase; AdoMet, S-adenosylmethionine; DTT, dithiothreitol; Pi, orthophosphate; PPi pyrophosphate, PPPi, tripolyphosphate; WT, wild type; PAGE, polyacrylamide gel electrophoresis.
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