|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 274, Issue 53, 37559-37564, December 31, 1999
From the Glycine N-methyltransferase (EC
2.1.1.20) catalyzes the methylation of glycine by
S-adenosylmethionine to form sarcosine and
S-adenosylhomocysteine. The enzyme was previously shown to be abundant in both the liver and pancreas of the rat, to consist of
four identical monomers, and to contain tightly bound folate polyglutamates in vivo. We now report that the inhibition
of glycine N-methyltransferase by
(6S)-5-CH3-H4PteGlu5 is
noncompetitive with regard to both S-adenosylmethionine and
glycine. The enzyme exhibits strong positive cooperativity with respect
to S-adenosylmethionine. Cooperativity increases with
increasing concentrations of
5-CH3-H4PteGlu5 and is greater at
physiological pH than at pH 9.0, the pH optimum. Under the same
conditions, cooperativity is much greater for the pancreatic form of
the enzyme. The Vmax for the liver form of the
enzyme is approximately twice that of the pancreatic enzyme, while
Km values for each substrate are similar in the liver and pancreatic enzymes. For the liver enzyme, at pH 7.0 half-maximal inhibition is seen at a concentration of about 0.2 µM
(6S)-5-CH3-H4PteGlu5,
while at pH 9.0 this value is increased to about 1 µM.
For the liver form of the enzyme, 50% inhibition with respect to
S-adenosylmethionine at pH 7.4 occurs at about 0.27 µM. The dissociation constant, Ks,
obtained from binding data at pH 7.4 is 0.095. About 1 mol of
(6S)-5-CH3-H4PteGlu5 was bound per tetramer at pH 7.0, and 1.6 mol were bound at pH 9.0. The
degree of binding and inhibition were closely parallel at each pH. At
equal concentrations of (6R,6S)- and
(6S)-5-CH3-H4PteGlu5, the natural (6S) form was about twice as inhibitory. These
studies indicate that glycine N-methyltransferase is a
highly allosteric enzyme, which is consistent with its role as a
regulator of methyl group metabolism in both the liver and the pancreas.
Glycine N-methyltransferase
(GNMT1; EC 2.1.1.20) carries
out the transfer of a methyl group to glycine from
S-adenosylmethionine (AdoMet) to form sarcosine and
S-adenosylhomocysteine (AdoHcy). It was first discovered by
Blumenstein and Williams (1) in guinea pig liver but was not purified.
Kerr (2) rediscovered the enzyme in crude extracts of rabbit liver when
the high activity of GNMT interfered with the measurement of RNA
methyltransferase. It was subsequently purified from rabbit liver (3)
and found to comprise 0.9-3% of the soluble protein.
GNMT is also very abundant in rat liver. It was purified by Ogawa and
Fujioka (4) and shown to consist of four identical subunits of
Mr = 32,423. We had earlier purified a
folate-binding protein from rat liver cytosol, which contained tightly,
but not covalently, bound
5-CH3-H4PteGlu5 in vivo
(5). This protein had a similar molecular weight, number of subunits,
and amino acid composition as rat liver GNMT. We subsequently showed
that this folate-binding protein was identical to GNMT (6). This was
surprising, since the folate does not participate in the enzyme reaction. Further studies showed, however, that when
5-CH3-H4PteGlu5 was bound, it
inhibited GNMT activity (7). We suggested that inhibition of GNMT by
5-CH3-H4PteGlu5 is a critical
component of a regulatory mechanism that links the availability of
preformed methyl groups (from dietary methionine) to the de
novo synthesis of methyl groups via the one-carbon folate pool (7,
8). Another abundant source of GNMT is the pancreas (2), and
5-CH3-H4PteGlu5 also acts as an
inhibitor of the pancreatic enzyme (9). More recently, the crystal
structure of recombinant rat liver GNMT has been resolved (10). The
binding of folates and their inhibition of GNMT were not investigated,
however. In this study, we characterize the binding of and inhibition
by 5-CH3-H4PteGlu5 to liver and pancreatic GNMT.
Materials--
Rat liver GNMT was purified from rat liver
extract by a modification of the method described previously by Suzuki
and Wagner (5). Rat pancreatic GNMT was purified from fresh pancreata as described by Yeo and Wagner (9). Synthetic folic acid pentaglutamate (PteGlu5) was provided by B. Schircks Laboratories, Jona,
Switzerland. Synthetic
(6R,6S)-5-CH3-H4PteGlu5
was prepared from the corresponding folate polyglutamates as described
by Yeo and Wagner (9). Synthetic (6S)-5-CH3-H4PteGlu5 was
prepared as described below. Bio-Safe II scintillation fluid was from
Research Products International Corp. AdoMet was obtained from Sigma,
and
S-adenosyl-L-[methyl-3H]methionine
(73.5 Ci/mmol) was from NEN Life Science Products. The labeled and
unlabeled AdoMet were repurified by a modification of the procedure by
Zappia et al. (11).
Synthesis of
(6S)-5-CH3-H4PteGlu5--
(6S)-5-CH3-H4PteGlu5
was prepared by a modification of the method used for the preparation
of radioactive (6S)-5-CH3-H4PteGlu from folic acid (12). The procedure consisted of direct reduction of
PteGlu5 to H4PteGlu5 by using
dihydrofolate reductase. The H4PteGlu5 reacted
with formaldehyde to give
5,10-methylene-H4PteGlu5, which was reduced to
5-CH3-H4PteGlu5 with sodium
borohydride. The details are described below.
About 0.25 µmol of PteGlu5 (100 µl) was added to the
reaction mixture containing 0.5 mg of NADPH (100 µl), 0.5 ml of 0.05 M sodium acetate buffer, pH 4.8, 9.9 mg of solid sodium
ascorbate, 10 µl of 0.37% (v/v) formaldehyde, and 0.3 ml of
distilled water. The solution was bubbled with N2 for 2 min, and then 1 mg of solid dihydrofolate reductase from
Lactobacillus casei (0.075 unit/mg, dry weight) was added.
It is important to use the enzyme from L. casei, because
dihydrofolate reductase from many other sources does not readily react
with PteGlun derivatives but only with 7, 8-H2PteGlun derivatives. The mixture was incubated under nitrogen in the dark for 1 h, at 37 °C. Additional
formaldehyde (250 µl of 0.37%) was added and incubated for an
additional 10 min. To the reaction mixture, 0.5 ml of cold deaerated
0.05 M Tris-HCl, pH 7.5, was added. Then 10 mg of sodium
borohydride in 100 µl of the Tris buffer was added in four portions
over a period of 5 min. The tube was flushed with nitrogen and
incubated at 37 °C for 1 h. After the incubation, the tube was
cooled in an ice bath, and 10 µl of 14 M
2-mercaptoethanol was added to protect folates from oxidation. The pH
was reduced to 5.0 with 5 N acetic acid to destroy excess
borohydride, and then the pH was adjusted back to 7.2 with 1 N NaOH. The product
((6S)-5-CH3-H4PteGlu5) was purified by fast protein liquid chromatography with a Mono Q
column, which was equilibrated with 10 mM 2-mercaptoethanol and was eluted with a gradient of 0-2 M ammonium acetate,
pH 7.2, in 10 mM 2-mercaptoethanol. The product was
lyophilized and taken up in 5 ml of 0.5 M potassium
phosphate, pH 7.0, and 40 mM 2-mercaptoethanol. Its
identity was confirmed by treatment with conjugase and high performance
liquid chromatography analysis as described by Wilson and Horne
(13).
Kinetic Studies--
Preliminary studies had shown that
inhibition of GNMT activity by
5-CH3-H4PteGlu5 was variable and
tended to be influenced by the order of addition of substrate or
inhibitor. When the enzyme was preincubated with
5-CH3-H4PteGlu5, inhibition was
consistently observed. Kinetic measurements were carried out using the
charcoal adsorption method for GNMT assay as described before (6) at pH
7.4 and at pH 9.0, the pH optimum. To examine the the effects of
varying glycine on GNMT activity, the reaction mixture contained about
1.5 µg of GNMT; either 0.2 M Hepes, pH 7.4, or 0.2 M Tris, pH 9.0; 0.5 mM
[3H-CH3]AdoMet (~5,000 dpm/nmol); and 5 mM DTT. To examine the effects of varying AdoMet, 8 mM glycine was included. Inhibition of activity was
determined by including
(6S)-5-CH3-H4PteGlu5 at
varying concentrations. The
5-CH3-H4PteGlu5 was made up in 40 mM 2-mercaptoethanol, and the same amount of
2-mercaptoethanol was added to the samples without inhibitor.
Measurement of inhibition was carried out by preincubating the enzyme,
buffer, DTT, and either
5-CH3-H4PteGlu5 or
2-mercaptoethanol for 15 min at 25 °C. The reaction was started by
the addition of substrates. The total volume of the incubation mixture
was 0.1 ml. Blank reactions were carried out in the absence of glycine.
Incubations were carried out at 25 °C for 15 min at pH 9.0 and for
30 min at pH 7.4. The reaction was stopped by the addition of 50 µl
of cold 10% trichloroacetic acid followed by 250 µl of a charcoal
suspension (38 mg/ml in 0.1 M acetic acid). After 15 min at
0 °C, the sample was centrifuged for 4 min in a microcentrifuge. Two
hundred µl of the supernatant was added to 5 ml of Bio-Safe II
scintillation fluid, and the radioactivity incorporated into sarcosine
was counted in a BetaTrac liquid scintillation system (Tm Analytic).
All assays were carried out in duplicate. Blank values obtained in the
absence of glycine were subtracted to give net counts. Enzyme activity
was expressed as nmol of product formed per min per mg of protein. The
data were analyzed using the Kaleidograph program (Synergy Software) to
fit the data to the Hill equation using nonlinear regression analysis.
Binding and Inhibition Studies--
Binding experiments were
carried out using liver GNMT and nonradioactive
(6S)-5-CH3-H4PteGlu5.
The components of the reaction mixture used to study binding were the
same as for measurement of enzyme activity, except that glycine and
AdoMet were omitted. For studies at pH 7.0, the reaction mixture
contained 0.1 M potassium phosphate, pH 7.0, 5 mM DTT, 0.35 µM GNMT, and various amounts of
(6S)-5-CH3-H4PteGlu5 in
a final volume of 300 µl. For studies at pH 9.0, the reaction mixture
was the same except that 0.2 M Tris-HCl, pH 9.0, buffer was
used. The amount of protein was calculated by the Bradford method (14)
after standardization of GNMT by amino acid analysis. Phosphate (0.1 M) was included in studies carried out at both pH values,
since the stock solution of the folate ligand was dissolved in 0.5 M, pH 7.0, phosphate. After incubation at 25 °C for 45 min, 200 µl of the reaction mixture was applied to an Amicon
micropartition system, MPS-1, containing a YMT ultrafiltration
membrane. The filter apparatus was centrifuged at 1000 × g for several minutes, and the unbound folate passed into
the filtrate. The free folate in the filtrate and the total folate in
the reaction mixture were measured by fluorescence. Bound folate was
determined by difference. Measurement of
5-CH3-H4PteGlu5 was carried out
fluorometrically using the method of Giulidori (15). The fluorescence
spectrometer (Perkin-Elmer, model 650-40) was set at 297 and 360 nm for
excitation and emission, respectively. Samples of 100 µl were
adjusted to pH 2.5, since the intensity of
5-CH3-H4PteGlu5 fluorescence is
much greater in acid. A blank was prepared using only enzyme, and
fluorescence values were corrected. A standard curve was prepared
using 5-CH3-H4PteGlu5. The amount of bound 5-CH3-H4PteGlu5/free
5-CH3-H4PteGlu5 was plotted
versus the concentration of bound
5-CH3-H4PteGlu5 in a Scatchard
plot, and maximum binding was determined by extrapolation. Companion inhibition studies were carried out by preincubating liver GNMT with
various concentrations of
(6S)-5-CH3-H4PteGlu5 for
15 min at 25 °C in a reaction mixture containing 0.2 M
Tris-HCl, pH 9.0, and 5 mM DTT in a volume of 60 µl. The
reaction mixture also contained 8 mM 2-mercaptoethanol and
0.1 M potassium phosphate, which were added with the folate
ligand. The enzyme reaction was started by bringing the volume to 100 µl with the addition of 0.2 µmol of glycine and 0.02 µmol of
[3H]AdoMet (4773 dpm/nmol). After a 30-min incubation at
25 °C, the reaction was stopped, and the enzyme activity was
measured as described above. Activity was compared with control samples preincubated in the absence of
(6S)-5-CH3-H4PteGlu5.
Inhibition of liver GNMT by
(6S)-5-CH3-H4PteGlu5 at
pH 7.0 was also examined. This was carried out in a 60-µl incubation
mixture with 0.1 M potassium phosphate buffer, pH 7.0, containing 4.5 µg of enzyme. After a 15-min preincubation at
25 °C, the substrates were added, and the volume was brought to 100 µl. The reaction was continued for 1 h.
Comparison of (6S)- and
(6R,6S)-5-CH3-H4PteGlu5 as
Inhibitors of GNMT--
Purified liver GNMT (1 µg) was preincubated
with 100 pmol of either
(6R,6S)-5-CH3-H4PteGlu5
or (6S)-5-CH3-H4PteGlu5
or without any addition in a solution containing 20 µmol of Tris-HCl,
pH 9.0, 0.5 µmol of DTT, 5 µmol of potassium phosphate, and 0.4 µmol of 2-mercaptoethanol in a final volume of 60 µl for 15 min.
The enzymatic reaction was started by the addition of
[3H]AdoMet and varying amounts of glycine, so the final
concentration of AdoMet was 0.5 mM and varying amounts of
glycine in a final volume of 0.1 ml. Incubation was carried out at
25 °C for 15 min. The reaction was stopped, and activity was
measured as described above.
Effect of 5-CH3-H4PteGlu5 on
Kinetic Parameters--
GNMT is a highly allosteric enzyme. Fig.
1 shows extensive cooperativity with
respect to AdoMet as substrate for the rat liver enzyme at pH 7.4. Cooperativity appears to be increased with increasing concentrations of
the inhibitory ligand,
5-CH3-H4PteGlu5. The pH optimum for
GNMT is 9.0 (4). At this pH, GNMT shows minimal cooperativity, but in
the presence of 5-CH3-H4PteGlu5
sigmoid kinetics is easily seen (Fig. 2).
In contrast, hyperbolic kinetics is seen with respect to the second
substrate, glycine, at both pH 7.4 and pH 9.0 (data not shown).
5-CH3-H4PteGlu5 behaves as a
noncompetitive inhibitor with respect to AdoMet. This is more clearly
seen at pH 9.0 (Fig. 3). Here, the data
are presented in a Hanes-Woolf plot to avoid the undue weighting of the
lowest concentration of substrate inherent in the double reciprocal
Lineweaver-Burk plot. It can be seen that three of the lines clearly
intersect on the x axis, while the fourth line is only
slightly below the others, indicating that the inhibition is
noncompetitive. The greater sigmoidicity at pH 7.4 results in plots
that are nonlinear and are difficult to interpret (data not shown). In
contrast, there is no cooperativity with respect to glycine as a
substrate, and a double reciprocal plot shows that
5-CH3-H4PteGlu5 is clearly a
noncompetitive inhibitor with respect to glycine (Fig.
4). Pancreatic GNMT is also inhibited by
5-CH3-H4PteGlu5. The pancreatic
enzyme appears to be even more allosteric than the liver enzyme,
although there is no significant cooperativity at pH 9.0 in the absence of 5-CH3-H4PteGlu5 (Fig.
5). The effects of
5-CH3-H4PteGlu5 on the
cooperativity of the liver and pancreatic forms of GNMT are summarized
as apparent Hill coefficients in Table I.
Values for Vmax(app) and
Km(app) are shown in Table
II. No data were collected for the
pancreatic enzyme at pH 7.4. The pancreatic enzyme is about half as
active as the liver enzyme under similar conditions.
Because reciprocal plots of velocities obtained with GNMT under
different concentrations of
5-CH3-H4PteGlu5 at pH 7.4 are not
linear, as with all enzymes exhibiting positive cooperativity, it was
impossible to determine the Ki from a Dixon plot. Therefore, the concentration of
5-CH3-H4PteGlu5 that results in 50% inhibition was determined at saturating (0.70 and 0.35 mM) concentrations of AdoMet. A value of about 0.27 µM was obtained (Fig. 6).
This value is comparable with the dissociation constant, Ks, obtained from binding data (see below).
The inhibitory effects of (6R,6S)- or
(6S)-5-CH3-H4PteGlu5 on
liver GNMT are shown in Fig. 7. When
equimolar amounts (1 µM) of either the
(R,S) or the (S) form were added, the
(S) form was approximately twice as inhibitory, suggesting
that the unnatural (R) form is a much less potent
inhibitor.
Inhibition and Binding by
(6S)-5-CH3-H4PteGlu5--
When
purified liver GNMT was incubated with increasing amounts of
(6S)-CH3-H4PteGlu5 for
1 h, complete inhibition was obtained at about 9 µM
at pH 7.0 (Fig. 8A).
Half-maximal inhibition occurred at about 0.2 µM. A
similar experiment was carried out at pH 9.0 (Fig. 8B), but
in this case the enzymatic reaction was continued for only 30 min. In
contrast, at pH 9.0, a maximum of 80% inhibition was obtained, and
half-maximal inhibition occurred at about 1.1 µM (Fig.
8B). The increased reaction time at pH 7.0 was required in
order to obtain accurate measurements, because GNMT is less active at
this pH. Binding studies were carried out in a similar manner but in
the absence of substrates. Following the incubation period, a portion
of the incubation mixture was rapidly filtered as described under
"Experimental Procedures" to separate bound and unbound folate. The
amount of 5-CH3-H4PteGlu5 was
measured by fluorescence after acidification as described under
"Experimental Procedures." Scatchard plots of the binding data
(Fig. 9) show that binding is concave
upward. Analysis of the data is complicated by the nonlinear nature of
the binding process, but extrapolation of the curves to the
x axis provides an estimate of the maximum concentrations of
ligand bound that are about 0.36 µM at pH 7.0 and 0.57 µM at pH 9.0. Since each experiment used 0.35 µM enzyme, this calculates to a stoichiometry of 1 mol of
5-CH3-H4PteGlu5 per tetramer at pH
7.0. At pH 9.0, the corresponding stoichiometry is 1.6. The binding
data were recalculated as percentage of maximum binding (% Bmax) at each concentration of
5-CH3-H4PteGlu5, and these values
were replotted in Fig. 8, A and B. There is a
very close correspondence between the amount of inhibition of GNMT activity and the amount of ligand bound, despite the fact that only 1 mol of bound 5-CH3-H4PteGlu5 causes
complete inhibition of the tetrameric enzyme at pH 7.0. At pH 9.0, the
binding of 1 mol of ligand produced about 80% inhibition. Hill plots
of the binding of 5-CH3-H4PteGlu5
at pH 9.0 and 7.0 gave n values of 0.65 and 1.0, respectively (data not shown). Values of n less than 1.0 and
Scatchard plots that are concave upward are characteristic of either a
heterogeneity of binding sites or negative cooperativity between
binding sites in which the first ligand has a higher apparent affinity
than subsequent ligands (16). Calculation of the dissociation constant
from the slopes of the extrapolated linear portions of the Scatchard
plots in Fig. 9, A and B, gave values of 0.095 µM at pH 7.4 and 1.9 µM at pH 9.0.
GNMT is believed to play a central role in the regulation of
methyl group metabolism of the liver (2). It is present in large
amounts, and the product of methyl transfer in the reaction is
sarcosine, which has no known physiologic role in mammals. For this
reason it has been suggested (2) that GNMT serves as a mechanism for
converting excess AdoMet, arising from methionine, to AdoHcy in order
to stabilize the ratio of AdoMet/AdoHcy. Since most methylation
reactions are product-inhibited by AdoHcy, this ratio is believed to
reflect the overall methylating ability of the cell (17). The fact that
GNMT is also inhibited by
5-CH3-H4PteGlu5 links the
availability of preformed methyl groups, as manifested by the level of
AdoMet, to the de novo synthesis of methyl groups via the
one-carbon folate pool (7). The abundance of GNMT in the pancreas as
well as the liver of the rabbit and the rat was first noted by Kerr
(2). Ogawa et al. (18) showed that mRNA for GNMT is
almost as abundant in pancreas as in liver. We have shown that
pancreatic GNMT contains tightly bound folate in vivo (9).
The present study shows that the patterns of inhibition of both liver
and pancreatic forms of GNMT are very similar.
The inhibition by 5-CH3-H4PteGlu5
has unusual features. It behaves in many respects as a slow, tightly
binding inhibitor. Preliminary studies indicated that inhibition was
consistent when the enzyme was preincubated with
5-CH3-H4PteGlu5 for 15 min before the addition of substrate. Because only a discontinuous assay for GNMT
activity is available, it has been impossible to describe the decrease
in reaction velocity as a function of time. Ogawa et al.
(19) have developed a continuous assay for GNMT activity that involves
coupling the production of S-adenosylhomocysteine to
S-adenosylhomocysteine hydrolase and adenosine deaminase.
The adenosine formed by S-adenosylhomocysteine hydrolase is
then converted to inosine with a decrease in absorbance at 265 nm.
Unfortunately, S-adenosylhomocysteine hydrolase is inhibited
by S-adenosylmethionine, and the coupled reaction cannot be
carried at concentrations of S-adenosylmethionine greater
than 0.1 mM.
5-CH3-H4PteGlu5 also binds very
tightly to GNMT. GNMT was first purified as a liver cytosolic
folate-binding protein. Radioactive folate was injected into rats, and
24 h later the liver cytosol contained a protein of about 130 kDa
that had tightly bound
5-CH3-H4PteGlu5 (20). In fact,
purification was dependent upon following the bound radioactivity over
several different columns. Only when passed over an anion exchange
column did the folate ligand dissociate (5). Tightly binding inhibitors
are effective at concentrations similar to that of the enzyme (21). In
the studies described here, GNMT was present at a concentration of
about 0.09 µM (for the tetrameric enzyme), and
CH3-H4PteGlu5 was present at
concentrations of 0.1-0.3 µM. Under these conditions, a
significant amount of enzyme and inhibitor should be present in an
essentially irreversible complex, and the assumptions upon which the
Michaelis-Menten and the Hill equations are based do not apply (22). It
is therefore surprising that the positive cooperativity of GNMT is
increased with increasing amounts of inhibitor. One would expect the
reaction rate to be decreased when significant amounts of enzyme are
tied up in an essentially irreversible complex but not that the
cooperativity of the enzyme would increase. Fig. 8A
indicates that at a concentration of 0.1 µM about 5% of
CH3-H4PteGlu5 was bound, and at 0.3 µM about 30% was bound at pH 7.4 (i.e. 95 and
70%, respectively, were unbound). It may be that the decreased amounts
of enzyme and inhibitor are not sufficient to significantly alter the
fit of the data to the Hill equation. Fig. 8A also shows
that it is possible to completely inhibit the enzyme, but this requires
far greater concentrations of
CH3-H4PteGlu5 (about 8 µM).
Ogawa and Fujioka (4) noted that liver GNMT exhibited positive
cooperativity with respect to AdoMet when measured at pH 7.4. It was
also stated that positive cooperativity was noted at pH 8.8. Interaction of AdoMet with the liver enzyme also showed positive
cooperativity when measured by the quenching of the intrinsic fluorescence at pH 7.4 (23) or by the binding of radioactive AdoMet at
pH 7.2 (24). Limited proteolysis of rat liver GNMT removed an 8-residue
N-terminal peptide, which changed the kinetics from sigmoid to
hyperbolic (24). The crystal structure of the recombinant rat liver
GNMT has recently been published (10). Crystals were grown at pH 6.5. The tetrameric structure shows the N terminus of each subunit deeply
associated with the other three subunits. We have not observed positive
cooperativity of the rat liver enzyme at pH 9.0 in the absence of
5-CH3-H4PteGlu5. This suggests that
the tetrameric enzyme is in a looser configuration at pH 9.0 than it is
at pH 7.0 and that the presence of the inhibitor causes a change in
conformation or association, which permits interaction among the
subunits. We have also obtained evidence that binding of
5-CH3-H4PteGlu5 results in a change
in the intrinsic fluorescence of
GNMT.2 Konishi and Fujioka
(24) showed that 4 mol of AdoMet were bound per tetramer, suggesting
the existence of four active sites. This has been confirmed by the
crystal structure, which revealed that AdoMet remained bound to each
monomer of the enzyme upon crystallization.
Inhibition of the liver enzyme by
5-CH3-H4PteGlu5 occurred at both pH
9.0 and 7.0. Binding and inhibition by
5-CH3-H4PteGlu5 closely paralleled
each other at both pH values (Fig. 8). Complete inhibition of activity
was observed at pH 7.0 when only 1 mol of ligand was bound per mol of
tetrameric enzyme. At pH 9.0, a maximum of 1.6 mol of ligand were bound
per tetramer. Fu et al. have suggested that
5-CH3-H4PteGlu5 binds at the AdoMet
binding site (10); however, this appears to be extremely unlikely.
Aside from the limited similarity between the pterin ring and the
purine ring, the two compounds are quite different in structure. AdoMet has a net positive charge, and the folate polyglutamate has multiple negative charges. AdoMet binds to four sites per tetramer and shows
positive cooperativity;
5-CH3-H4PteGlu5 binds to one site per tetramer and shows negative cooperativity. It is therefore possible
that 5-CH3-H4PteGlu5 binds to a
region that is composed of contributions by more than one subunit. The
binding of 5-CH3-H4PteGlu5 may then
restrict access of AdoMet to its binding sites. These suggestions
should be clarified by locating
5-CH3-H4PteGlu5 within the crystal
structure of the molecule.
The concentration of
5-CH3-H4PteGlu5 in rat liver has
been estimated to be about 9.1 µM (25). From the same
reference, the concentration of AdoMet can be estimated to be about 0.4 mM. The apparent Km for AdoMet is about
0.39 mM (liver enzyme; Table II). Complete inhibition
occurs at 10 µM
5-CH3-H4PteGlu5 (Fig.
8A), and 50% inhibition occurs at about 0.27 µM (Fig. 6). Therefore, the concentrations of these
compounds in the liver are in the range where changes could modulate
the activity of the enzyme.
The complete inhibition of a tetrameric enzyme containing four active
sites by a single mole of inhibitor is highly unusual. It would be of
benefit, however, for the regulation of GNMT under physiologic
conditions, because the enzyme activity can be completely turned on or
off through a relatively narrow concentration range of
5-CH3-H4PteGlu5. If there were four
sites for binding and inhibition, then complete inhibition would
require 4 times as much
5-CH3-H4PteGlu5, much more than is
actually present (25).
The binding of 5-CH3-H4PteGlu5 to
GNMT is similar in many aspects to the binding of folate polyglutamates
to deoxyhemoglobin. Benesch and co-workers (26, 27) showed that folate
polyglutamates are bound to deoxyhemoglobin tetramers at a site to
which 2,3-diphosphoglycerate is also bound. Only a single molecule of
the folate polyglutamate was bound to the deoxyhemoglobin tetramer.
Both 5-CH3-H4PteGlu7 and
PteGlu7 as well as the polyglutamate form of methotrexate were bound with dissociation constants ranging from 0.025 to 0.14 mM at a site that involves the central cavity of the
deoxyhemoglobin tetramer. In oxyhemoglobin, the central cavity is too
small to accommodate the folate polyglutamate (28). It is tempting to think that 5-CH3-H4PteGlu5 may bind
to the GNMT tetramer in a similar manner. The availability of the
crystal structure of the enzyme makes such studies now possible.
We acknowledge Byron Glenn and W. Decha-Umphai for excellent technical assistance. In addition, we thank
Drs. D. W. Horne and R. J. Cook for useful discussion on
methodology. We also are grateful to Dr. R. L. Kisliuk for
discussion on the interaction of folate polyglutamate with GNMT.
*
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.
§
Present address: Dept. of Biochemistry, College of Medicine, Cheju
National University, 1 Ara 1-dong, Cheju, Cheju-do, 690-756, Republic
of Korea.
2
E.-J. Yeo, W. T. Briggs, and C. Wagner,
unpublished results.
The abbreviations used are:
GNMT, glycine
N-methyltransferase;
AdoMet, S-adenosylmethionine;
AdoHcy, S-adenosylhomocysteine;
PteGlu5, folic acid
pentaglutamate;
H2PteGlun, dihydrofolate
polyglutamate;
H4PteGlu5, tetrahydrofolate
pentaglutamate;
5-CH3-H4PteGlu5, 5-methyltetrahydrofolate pentaglutamate;
DTT, dithiothreitol.
Inhibition of Glycine N-Methyltransferase by
5-Methyltetrahydrofolate Pentaglutamate*
§,¶
Department of Biochemistry, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232-0146 and the
¶ Department of Veterans Affairs Medical Center,
Nashville, Tennessee 37212
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (18K):
[in a new window]
Fig. 1.
Effect of AdoMet concentration on liver GNMT
activity at pH 7.4 in the absence and presence of
(6S)-5-CH3-H4PteGlu5.
GNMT was present at 0.09 µM. AdoMet concentration was
varied from 0.02 to 0.7 mM. Glycine was maintained at 8 mM. Velocity is expressed as nmol of product formed/min/mg
of protein. Other details are described under "Experimental
Procedures."
View larger version (20K):
[in a new window]
Fig. 2.
Effect of AdoMet concentration on liver GNMT
activity at pH 9.0 in the absence and presence of
(6S)-5-CH3-H4PteGlu5.
Conditions were the same as for Fig. 1 except that the pH was 9.0 and
incubation was for 15 min.
View larger version (17K):
[in a new window]
Fig. 3.
Hanes-Woolf plot of the effect of AdoMet
concentration on liver GNMT activity at pH 9.0 in the absence and
presence of
(6S)-5-CH3-H4PteGlu5.
Conditions were the same as for Fig. 2.
View larger version (14K):
[in a new window]
Fig. 4.
Double reciprocal plot of the effect of
glycine concentration on liver GNMT activity at pH 9.0 in the absence
and presence of 0.3 µM
(6R,6S)-5-CH3-H4PteGlu5.
AdoMet was maintained at 0.5 mM, and glycine was varied
from 0.1 to 4.0 mM. All other conditions were the same as
for Fig. 2.
View larger version (19K):
[in a new window]
Fig. 5.
Effect of AdoMet concentration on pancreatic
GNMT activity at pH 9.0 in the absence and presence of
(6S)-5-CH3-H4PteGlu5.
Conditions were the same as for Fig. 2.
Cooperativity of glycine N-methyltransferase
Kinetic parameters for liver and pancreatic GNMT
View larger version (15K):
[in a new window]
Fig. 6.
50% inhibition of
(6R,6S)-5-CH3-H4PteGlu5
on liver GNMT at pH 7.4. Conditions were the same as in Fig.
1.
View larger version (14K):
[in a new window]
Fig. 7.
Inhibition of liver GNMT at pH 9.0 by
(6S) and
(6R,6S)-5-CH3-H4PteGlu5.
The concentration of (6S)- or
(6R,6S)-5-CH3-H4PteGlu5
was 1 µM. GNMT was present at 0.06 µM.
Other details are described under "Experimental Procedures."
View larger version (13K):
[in a new window]
Fig. 8.
Inhibition by and binding of
(6S)-5-CH3-H4PteGlu5
to liver GNMT. The experiment shown in A was carried
out at pH 7.0, and that shown in B was carried out at pH
9.0. Measurement of enzyme activity is described under "Experimental
Procedures" and is expressed as percentage of inhibition. GNMT was
present at 0.28 µM. Measurement of binding was carried
out as described under "Experimental Procedures." The data were
taken from Fig. 9 and replotted as percentage of maximum binding.
Open circles, percentage of
Bmax; closed circles,
percentage of inhibition.
View larger version (12K):
[in a new window]
Fig. 9.
Scatchard plot of the specific binding of
(6S)-5-CH3-H4PteGlu5 to liver
GNMT. A, purified enzyme (14 µg) was incubated with
various concentrations of
(6S)-5-CH3-H4PteGlu5 for
75 min at 25 °C in a total volume of 300 µl at pH 7.0 to provide a
final enzyme concentration of 0.35 µM. Total and free
5-CH3-H4PteGlu5 was determined as
described under "Experimental Procedures." B, purified
enzyme (14 µg) was incubated with various concentrations of
(6S)-5-CH3-H4PteGlu5 for
45 min in a total volume of 300 µl at pH 9.0 to provide a final
enzyme concentration of 0.35 µM. Other details were as
described above and under "Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence and reprint requests should be
addressed: Vanderbilt University School of Medicine, 620 Light Hall, Nashville, TN 37232-0146. Tel.: 615-343-9866; Fax: 615-343-0704.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Blumenstein, J.,
and Williams, G. R.
(1960)
Biochem. Biophys. Res. Commun.
3,
259-263[CrossRef]
2.
Kerr, S. J.
(1972)
J. Biol. Chem.
247,
4248-4252 3.
Heady, J. F.,
and Kerr, S. J.
(1973)
J. Biol. Chem.
248,
69-72 4.
Ogawa, H.,
and Fujioka, M.
(1982)
J. Biol Chem.
257,
3447-3452 5.
Suzuki, H.,
and Wagner, C.
(1980)
Arch. Biochem. Biophys.
199,
236-248[CrossRef][Medline]
[Order article via Infotrieve]
6.
Cook, R. J.,
and Wagner, C.
(1984)
Proc. Natl. Acad. Sci. U. S. A.
81,
3631-3634 7.
Wagner, C.,
Briggs, W. T.,
and Cook, R. J.
(1985)
Biochem. Biophys. Res. Commun.
127,
746-752[CrossRef][Medline]
[Order article via Infotrieve]
8.
Balaghi, M.,
and Wagner, C.
(1992)
J. Nutr.
122,
1391-1396
9.
Yeo, E.,
and Wagner, C.
(1992)
J. Biol. Chem.
267,
24669-24674 10.
Fu, Z.,
Hu, Y.,
Konishi, K.,
Takata, Y.,
Ogawa, H.,
Gomi, T.,
Fujioka, M.,
and Takasugawa, F.
(1996)
Biochemistry
35,
11985-11993[CrossRef][Medline]
[Order article via Infotrieve]
11.
Zappia, V.,
Carteni-Farina, M.,
Calleti, P.,
Ragione, F. D.,
and Cacciapnoti, G.
(1983)
Methods Enzymol.
94,
57-66[Medline]
[Order article via Infotrieve]
12.
Horne, D. W.,
Briggs, W. T.,
and Wagner, C.
(1980)
Methods Enzymol.
66,
545-547[Medline]
[Order article via Infotrieve]
13.
Wilson, S. D.,
and Horne, D. W.
(1986)
Methods Enzymol.
122,
269-273[Medline]
[Order article via Infotrieve]
14.
Bradford, M. M.
(1976)
Anal. Biochem.
72,
248-254[CrossRef][Medline]
[Order article via Infotrieve]
15.
Giulidori, P.,
Galli-Kienle, M.,
and Stramentinoli, G.
(1981)
Clin. Chem.
27,
2041-2043 16.
Dahlquist, F. H.
(1978)
Methods Enzymol.
48,
270-299[Medline]
[Order article via Infotrieve]
17.
Cantoni, G. L.,
Richards, H. H.,
and Chiang, P. K.
(1978)
in
Transmethylation
(Usdin, E.
, Borchardt, R. T.
, and Creveling, C. R., eds)
, pp. 155-164, Elsevier Science Publishing Co., Inc., New York
18.
Ogawa, H.,
Gomi, T.,
Horii, T.,
Ogawa, H.,
and Fujioka, M.
(1984)
Biochem. Biophys. Res. Commun.
124,
44-50[Medline]
[Order article via Infotrieve]
19.
Ogawa, H.,
Gomi, T.,
Takata, Y.,
Date, T.,
and Fujioka, M.
(1997)
Biochem. J.
327,
407-412
20.
Zamierowski, M. M.,
and Wagner, C.
(1977)
J. Biol. Chem.
252,
933-936 21.
Morrison, J. F.
(1969)
Biochim. Biophys. Acta
185,
269-286[Medline]
[Order article via Infotrieve]
22.
Segel, I. H.
(1993)
Enzyme Kinetics: Wiley Classics Library Edition
, pp. 15-151, John Wiley & Sons, Inc., New York
23.
Konishi, K.,
and Fujioka, M.
(1987)
Biochemistry
26,
8496-8502[Medline]
[Order article via Infotrieve]
24.
Konishi, K.,
and Fujioka, M.
(1988)
J. Biol. Chem.
263,
13381-13385 25.
Horne, D. W.
(1989)
BioFactors
2,
65-68[Medline]
[Order article via Infotrieve]
26.
Benesch, R. E.,
Benesch, R.,
Kwong, S.,
and Baugh, C. M.
(1983)
Proc. Natl. Acad. Sci. U. S. A.
80,
6202-6205 27.
Benesch, R. E.,
Kwong, S.,
Benesch, R.,
and Baugh, C. M.
(1985)
J. Biol. Chem.
260,
14653-14658 28.
Arnone, A.,
Rogers, P. H.,
Benesch, R. E.,
Benesch, R.,
and Kwong, S.
(1986)
J. Biol. Chem.
261,
5853-5857
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  Z. Luka, S. Pakhomova, L. V. Loukachevitch, M. Egli, M. E. Newcomer, and C. Wagner 5-Methyltetrahydrofolate Is Bound in Intersubunit Areas of Rat Liver Folate-binding Protein Glycine N-Methyltransferase J. Biol. Chem., February 9, 2007; 282(6): 4069 - 4075. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Field, D. M. E. Szebenyi, and P. J. Stover Regulation of de Novo Purine Biosynthesis by Methenyltetrahydrofolate Synthetase in Neuroblastoma J. Biol. Chem., February 17, 2006; 281(7): 4215 - 4221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Beagle, T. L. Yang, J. Hung, E. A. Cogger, D. J. Moriarty, and M. A. Caudill The Glycine N-Methyltransferase (GNMT) 1289 C->T Variant Influences Plasma Total Homocysteine Concentrations in Young Women after Restricting Folate Intake J. Nutr., December 1, 2005; 135(12): 2780 - 2785. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Ulrey, L. Liu, L. G. Andrews, and T. O. Tollefsbol The impact of metabolism on DNA methylation Hum. Mol. Genet., April 15, 2005; 14(suppl_1): R139 - R147. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Goyer, T. L. Johnson, L. J. Olsen, E. Collakova, Y. Shachar-Hill, D. Rhodes, and A. D. Hanson Characterization and Metabolic Function of a Peroxisomal Sarcosine and Pipecolate Oxidase from Arabidopsis J. Biol. Chem., April 23, 2004; 279(17): 16947 - 16953. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Herbig, E.-P. Chiang, L.-R. Lee, J. Hills, B. Shane, and P. J. Stover Cytoplasmic Serine Hydroxymethyltransferase Mediates Competition between Folate-dependent Deoxyribonucleotide and S-Adenosylmethionine Biosyntheses J. Biol. Chem., October 4, 2002; 277(41): 38381 - 38389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Rowling, M. H. McMullen, D. C. Chipman, and K. L. Schalinske Hepatic Glycine N-Methyltransferase Is Up-Regulated by Excess Dietary Methionine in Rats J. Nutr., September 1, 2002; 132(9): 2545 - 2550. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |