(Received for publication, April 17, 1995)
From the
5,10-Methenyltetrahydrofolate synthetase catalyzes the
irreversible conversion of 5-formyl-tetrahydropteroylpolyglutamates
(5-CHO-HPteGlu
) to
5,10-methenyltetrahydropteroylpolyglutamates (5,
10-CH
-H
PteGlu
). The
equilibrium of the nonenzymatic reaction, which equilibrates slowly in
the absence of enzyme, greatly favors
5-CHO-H
PteGlu
. The enzyme couples the
reaction to the hydrolysis of ATP shifting the equilibrium to favor
5,10-CH
-H
PteGlu
.
Substrate-dependent non-equilibrium isotope exchange of
ADP into ATP was observed, suggesting the
formation of a phosphorylated intermediate of
5-CHO-H
PteGlu
during the
enzyme-catalyzed reaction. The competitive inhibitor
5-formyltetrahydrohomofolate also supported the ADP to ATP exchange,
suggesting that this molecule could also form a phosphorylated
intermediate. The initial rates of the ADP-ATP exchange with saturating
ADP were about 70 s
for both compounds, while the k
values for product formation were 5
s
for 5-CHO-H
PteGlu
and 0.005 s
for
5-formyltetrahydrohomofolate. Starting with
5-[
O]CHO-H
PteGlu
,
it was shown by
P NMR that the formyl oxygen of the
substrate was transferred to the product phosphate during the reaction.
This further supports the existence of a phosphorylated intermediate.
The formyl group of 5-CHO-H
PteGlu
is
known to be an equilibrium mixture of two rotamers. Stopped-flow
analysis of the enzymatic reaction showed that only one of the rotamers
serves as a substrate for the enzyme.
5,10-Methenyltetrahydrofolate synthetase (MS) ()(EC
6.3.3.2), also referred to as 5-formyltetrahydrofolate cyclodehydrase,
catalyzes the ATP-dependent conversion of 5-formyltetrahydrofolate
(5-CHO-H
PteGlu
) to
5,10-methenyltetrahydrofolate
(5,10-CH
-H
PteGlu
) (). The enzyme activity was first found in sheep liver
acetone powder extracts by Peters and Greenberg (1) and later
studied by Kay et al. and Greenberg et
al.(2, 3) . This enzyme has been purified to
homogeneity from both prokaryotic and eukaryotic sources (4, 5, 6) . The primary structure of the
enzyme from rabbit liver has been determined(7) .
The origin and function of 5-CHO-HPteGlu
has been a source of confusion and debate(8) . The
acid-catalyzed interconversion of 5-CHO-H
PteGlu
and 5,10-CH
-H
PteGlu
occurs nonenzymatically with the equilibrium greatly
favoring 5-CHO-H
PteGlu
at pH
7(2, 9) . The rate of the nonenzymatic reaction occurs
on the time scale of hours under physiological conditions and is
complicated by 5,10-CH
-H
PteGlu
also being hydrolyzed to
10-CHO-H
PteGlu
. By coupling to ATP hydrolysis, the equilibrium is shifted to favor
5,10-CH
-H
PteGlu
at pH
7. For many years, neither an enzymatic source nor function of
5-CHO-H
PteGlu
was known. This
suggested that the function of MS was to serve as a salvage pathway for
the reincorporation of this nonenzymatically produced folate derivative
into the one-carbon pool.
5-CHO-HPteGlu
has been found to occur in many cells at low
concentrations(8) . However, its presence in cells is suspect
since the harsh procedures used to extract folates are known to convert
10-CHO-H
PteGlu
to
5-CHO-H
PteGlu
. Recently, it was shown
that in Neurospora crassa conidiospores at least 85% of the
folate pool is
5-CHO-H
PteGlu
(10) . The mild
extraction procedure was shown not to account for the presence of the
5-CHO-H
PteGlu
. Because
5-CHO-H
PteGlu
is the only
tetrahydrofolate derivative that is stable to oxidative degradation,
these studies suggested that a physiological role is to serve as a
storage form of this coenzyme in dormant stages of cellular life
cycles. It has also been noted that 5-CHO-H
PteGlu
is an inhibitor of many enzymes in one-carbon metabolism,
suggesting it may also serve a regulation role(8) . Making irreversible would be beneficial to a cell whether
5-CHO-H
PteGlu
is formed
nonenzymatically, is used as a storage form for folates, or functions
in regulation by inhibiting enzymes in one-carbon metabolism.
5-CHOHPteGlu, clinically known as leucovorin, has been
administered to cancer patients to rescue them from toxicity during
high dose methotrexate chemotherapy, or more recently to enhance the
cytotoxic activity of 5-fluorouracil(11, 12) . MS is
the only known enzyme that utilizes 5-CHO-H
PteGlu
as a substrate. For 5-CHO-H
PteGlu to be
effective as a rescue agent and an enhancer of 5-fluorouracil toxicity,
it must first be converted to
5,10-CH
-H
PteGlu through MS activity. The
effectiveness of leucovorin in chemotherapy is at least partially
dependent on the irreversibility of . It has been shown
that doubling intracellular concentrations of
5-CHO-H
PteGlu
, by inhibiting MS
activity, suppressed growth of MCF-7 human breast cells by
80%(13) . This suggests that MS could be a potentially
important enzyme as a target in chemotherapy.
In this paper, we
report the mechanism of the coupling of ATP hydrolysis to the
conversion of 5-CHO-HPteGlu
to
5,10-CH
-H
PteGlu
by
MS. By using NMR spectroscopy and isotope exchange techniques, a
5-CHO-H
PteGlu
phosphorylated
intermediate was demonstrated. The role of this intermediate in making irreversible is discussed.
To determine reversibility of , a 1-ml reaction solution containing 0.5 mM [H]MgADP (2 µCi) and either 200
µM 5,10-CH
-H
PteGlu or 100
µM 5,10-CH
-H
PteGlu
was incubated with 7.2 µg of MS in 50 mM potassium
phosphate, pH 7.2, and 100-µl aliquots were removed at 30, 60, and
90 min, which were treated the same as described above except that 20
µl of a 2.5 mM MgATP solution was added to the extract as
an ATP carrier before injection into the HPLC.
The
affinity of 5-CHO-tetrahydrohomofolate for MS was determined by
observing it as a competitive inhibitor of 5-CHO-H4PteGlu. Using only
the monoglutamate form of the substrate and 5-CHO-tetrahydrohomofolate,
a competitive inhibition pattern was observed in a double reciprocal
plot of A
versus 5-CHO-H
PteGlu concentration. A K
of 0.1 µM was determined. This shows that
5-CHO-tetrahydrohomofolate has a high affinity for the active site of
MS but exhibits a low k
value.
Figure 1:
Substrate-dependent incorporation of
ADP into ATP. A 0.5-ml solution containing 1 mM MgATP, 0.5
mM [H]MgADP (5 µCi), 2 µg of MS,
and either 2 mM (6R,6S)-5-CHO-H
PteGlu or 28
µM 5-CHO-tetrahydrohomofolate was incubated in 50 mM KMES, pH 6.0, at 30 °C. Aliquots at 30-120 min were
removed and analyzed for formation of [
H]ATP as
described under ``Experimental Procedures.'' The circles and squares represent the nmol of
[
H]ATP formed in the presence of
5-CHO-H
PteGlu or 5-CHO-tetrahydrohomofolate, respectively.
Controls containing no enzyme gave about 200 cpm per aliquot, and the
reaction solutions ranged in values from 1500 to 24,000
cpm.
The exchange experiments were
also performed with increasing concentrations of MgADP at saturating
levels of 5-CHO-HPteGlu. The first 30 min of radiolabel
incorporation into ATP were measured. The results show that the
conversion of ADP to ATP depends on the initial concentration of ADP (Fig. 2). A double reciprocal plot of initial rate of
[
H]ATP formation versus the
concentration of ADP is linear, giving an apparent K
of 1.4 mM for ADP and a k
for the
formation of ATP of 70 s
. A product inhibition
constant of 0.3 mM for ADP has been previously
determined(7) .
Figure 2:
Dependence of the ADP-ATP exchange rate on
ADP concentration. A 0.5-ml reaction containing 1 mM MgATP, 2
mM (6R,6S)-5-CHO-HPteGlu, 2 µg of MS, and
varied amounts of [
H]MgADP (0.25, 0.5, 1.0, and
2.0 mM) in KMES, pH 6.0, were incubated at 30 °C for 30
min. Aliquots were removed and analyzed for
[
H]ATP as described under ``Experimental
Procedures.'' The inset represents a Lineweaver-Burk plot
of the rate of [
H]ATP formation
(µM/min) versus the initial concentration of
MgADP (mM).
Figure 3:
[P] NMR analysis of
inorganic phosphate from a reaction starting with
5-CH[
O]-H
PteGlu
and
MgATP. A reaction solution containing 5 mM MgATP and 64%
enriched 5-CH[
O]-H
PteGlu
in 20 mM KMES, pH 6.0, was placed in an NMR tube and
analyzed for
P-labeled inorganic phosphate (panelA). To this solution was added 2.4 µg of MS, and
after 1 h another spectrum was obtained for
P-labeled
inorganic phosphate (panelB). The phosphate peak in panelA is residual phosphate from the substrate
solution. The double peak in panelB represents the
formation of one equivalent of phosphate based on the initial
concentration of 5-CHO-H
PteGlu
. After
subtracting the residual phosphate peak from panelA,
the newly formed peaks in panelB represent 34%
[O
]phosphate and 66% singly labeled
[O
]phosphate.
If only one rotamer of 5-CHO-HPteGlu
serves as a substrate for MS, the presence of excess enzyme should
rapidly convert this rotamer to
5,10-CH
-H
PteGlu with conversion of the
second rotamer being controlled by the much slower nonenzymatic
interconversion of the two rotamers. Using stopped-flow
spectrophotometry, the rate of product formation can be followed with
the use of excess enzyme. Fig. 4shows the rate of
5,10-CH
-H
PteGlu
formation at
360 nm when 11.6 µM MS was flowed against 11.7 µM 5-CHO-H
PteGlu
and 1.9 mM MgATP at
21 °C. The data were fitted to a double exponential equation as
represented by the solidline. The slower rate was
0.03 s
and was the same when either
5-CHO-H
PteGlu or 5-CHO-H
PteGlu
was
used as substrate. This rate is, within experimental error, the same
rate of rotamer interconversion previously determined on our
instrument(25) . The ratio of the amplitudes for the rapid and
slow phases was 2.5. This result is also very close to the equilibrium
ratio (2.35:1 at 25 °C) determined by NMR
spectroscopy(23) . These results suggest that MS uses as the
substrate only the more abundant rotamer of both
5-CHO-H
PteGlu and 5-CHO-H
PteGlu
.
This is the same rotamer bound by dihydrofolate reductase (24) and serine hydroxymethyltransferase(25) .
Figure 4:
Rate of formation of
5,10-CH-H
PteGlu
with excess MS
as analyzed by stopped-flow spectrophotometry. A solution of 11.6
µM MS in 20 mM KMES, pH 6.0, was flowed against
11.7 µM 5-CHO-H
PteGlu
and 1.9
mM MgATP solution at 21 °C. The solidline is a curve fit for a two-exponential reaction. The inset is expanded data from the first 2 s of the
reaction.
The metabolite
5,10-CH-H
PteGlu
can be
hydrolyzed to both 10-CHO-H
PteGlu
and
5-CHO-H
PteGlu
at neutral pH. Its K
for formation of
10-CHO-H
PteGlu
can be readily determined
experimentally, but the K
for formation of
5-CHO-H
PteGlu
can only be determined under
acidic conditions and is less accurate. Using the K
experimentally determined by Kay et al.(2) for , we calculate that the equilibrium ratio of product to
reactant at pH 7.0 is 1:6.5
10
.
Coupling ATP hydrolysis to results in ()with a predicted K
` of
66 M. A similar calculation, based on the
tetrahydroquinoxaline analog of 5-CHO-H
PteGlu
,
indicates that the K
` for would
approach 500 M(2, 9) . Our failure to find
any incorporation of labeled ADP into ATP puts an upper limit on the
rate of the reverse of of 0.08 min
.
This is nearly 4000-fold slower than the enzyme-catalyzed rate of the
forward reaction. This suggests that at pH 7.0 the equilibrium of does not favor 5-CHO-H
PteGlu
to
the extent predicted by the studies of Kay et al., since the
predicted K
` of 66 M for would show some isotope exchange of ADP into ATP.
The
studies of non-equilibrium isotope exchange of ADP and ATP with both
5-CHO-HPteGlu and 5-CHO-tetrahydrohomofolate suggest that
although the overall reaction is irreversible, there is a reversible
step involved on the pathway to the formation of
5,10-CH
-H
PteGlu
. Fig. S1proposes a mechanism to explain the observed results. The
major rotamer of the enolate form of 5-CHO-H
PteGlu (structureI) makes a nucleophilic attack on the
-phosphoryl group of ATP to form a phosphorylated
5-CHO-H
PteGlu (structureII, Fig. S1). At this intermediate, enzyme-bound ADP can freely
equilibrate with solvent ADP. Reversal of this reaction (II to I) would
account for the incorporation of [
H]ADP into ATP (Fig. 1). Intermediate II would be formed with both
5-CHO-H
PteGlu and 5-CHO-tetrahydrohomofolate. The studies
with varied amounts of ADP show saturation kinetics (Fig. 2).
The linear double reciprocal plot suggests a Michaelis-Menten mechanism
in which the K
for ADP is 1.4 mM and the
rate from ADP to ATP is about 70 s
. This rate is
essentially the same rate determined from the study of ADP and ATP
isotope exchange in the presence of 5-CHO-tetrahydrohomofolate (66
s
). The rate of conversion of intermediate II to I
is 10 times the k
of 5 s
determined previously for (6) .
Figure S1: Scheme IProposed mechanism for 5,10-methenyltetrahydrofolate synthetase.
Intermediate II can proceed further in the reaction by attack of
N of 5-CHO-H
PteGlu to form a putative
tetrahedral intermediate (structureIII, Fig. S1). This would collapse to eliminate phosphate and form
the product 5,10-CH
-H
PteGlu (structureIV). It would be this last step that may be essentially
irreversible since phosphate would be a poor nucleophile in the back
reaction. The value of k
probably is determined
by either the conversion of intermediate II to III or the elimination
of phosphate to form product. The observation that k
for 5-CHO-tetrahydrohomofolate is 1000-fold less than the k
for 5-CHO-H
PteGlu suggests that it
is the conversion of intermediate II to intermediate III. The N
of the homofolate analog would probably be out of optimum
position to make the nucleophilic attack on the phosphorylated
intermediate II.
The NMR study provides additional evidence for the
formation of the phosphorylated intermediate II. The mechanism in Fig. S1predicts that the formyl oxygen of
5-CHO-HPteGlu would be quantitatively transferred to
phosphate. Starting the reaction with
5-[
O]CHO-H
PteGlu, NMR analysis shows
that the oxygen is transferred to phosphate. An alternative mechanism,
which would be consistent with the NMR data, is that ATP forms a
phosphorylated enzyme intermediate and that 5-CHO-H
PteGlu
is phosphorylated by the phosphoenzyme. However, this mechanism
suggests that the enzyme would catalyze the equilibration of
[
H]ADP and ATP in the absence of the
5-CHO-H
PteGlu. We found no evidence for this
substrate-independent ADP-ATP exchange, which argues against a
phosphorylated enzyme being formed.
Another folate-dependent enzyme
that utilizes ATP is N-formyltetrahydrofolate synthetase,
which catalyzes the synthesis of 10-formyltetrahydrofolate from formate
and tetrahydrofolate. ATP hydrolysis to ADP and phosphate is coupled to
this reaction to shift the equilibrium to favor
10-CHO-H
PteGlu. In eukaryotic cells, this activity is part
of a trifunctional enzyme, while in bacteria it is
monofunctional(26) . Mejillano et al.(26) demonstrated that both the prokaryotic and eukaryotic
enzymes form a formyl phosphate as an intermediate. However, ADP
remains bound to the enzyme so the formate-dependent ADP-ATP exchange
is very slow. Schrimsher et al.(27) have shown that
aminoimidazole ribonucleotide synthetase catalyzes the ATP-dependent
formation of the imidazole ring in purine biosynthesis. Starting with
[
O]formylglycinamidine ribonucleotide, these
authors showed the existence of a phosphorylated intermediate of the
substrate. However, ADP-ATP exchange was only 1/20th the rate of the
reaction, again probably the result of a slow release of ADP from the
enzyme.
Addendum-During the review of this
manuscript, a communication by Kounga et al.(28) was
published, which presented similar P NMR data as shown in Fig. 3.