(Received for publication, June 7, 1995; and in revised form, July 31, 1995)
From the
GMP synthetase (EC 6.3.5.2) is an amidotransferase that
catalyzes the amination of xanthosine 5`-monophosphate to form GMP in
the presence of glutamine and ATP. Glutamine hydrolysis produces the
necessary amino group while ATP hydrolysis drives the reaction. Ammonia
can also serve as an amino group donor. GMP synthetase contains two
functional domains, which are well coordinated. The ``glutamine
amide transfer'' or glutaminase domain is responsible for
glutamine hydrolysis. The synthetase domain is responsible for ATP
hydrolysis and GMP formation. Inorganic pyrophosphate inhibits the
synthetase and uncouples the two domain functions by allowing glutamine
hydrolysis to take place in the absence of ATP hydrolysis or GMP
formation. Acivicin, a glutamine analog, selectively abolishes the
glutaminase activity. It inhibits the synthetase activity only when
glutamine is the amino donor. When ammonia is used in place of
glutamine, acivicin has no effect on the synthetase activity. Acivicin
inhibits GMP synthetase irreversibly by covalent modification. Enzyme
inactivation is greatly facilitated by the presence of substrates.
Acivicin labels GMP synthetase at a single site, and a tryptic peptide
containing the modified residue was isolated. Mass spectrometry and
Edman sequence analysis show that Cys is the site of
modification. This residue is conserved among GMP synthetases and is
located within a predicted glutamine amide transfer domain. These data
suggest that Cys
is an essential residue involved in the
hydrolysis of glutamine to produce an amino group and is not needed for
the hydrolysis of ATP or amination of xanthosine 5`-monophosphate to
produce GMP.
The amidotransferases are a group of biosynthetic enzymes that
incorporate nitrogen atoms into a variety of metabolites. These enzymes
are constructed with two domains of distinct functions. The GAT ()(or glutaminase) function is responsible for utilizing
glutamine to produce the nitrogen source. The synthetase function is
responsible for using the nitrogen source produced from glutamine
hydrolysis in the amination of the nitrogen-accepting metabolite (for a
review of amidotransferases, see (1) ).
GMP synthetase is a member of the G-type amidotransferase family and catalyzes the amination of XMP to produce GMP in the de novo synthesis of guanine nucleotides. The designation of ``G-type'' is based on sequence similarities with the trpG-encoded anthranilate synthase component II. Similar to other family members, GMP synthetase can utilize either ammonia or glutamine as a source of nitrogen(2, 3, 4, 5) . During catalysis, glutamine is hydrolyzed to generate the amino group needed for the amination reaction. In the presence of magnesium ion, ATP drives the reaction. GMP synthetase is unique among the G-type amidotransferases in that it hydrolyzes ATP to AMP and inorganic pyrophosphate. The other ATP-utilizing family members produce ADP and inorganic phosphate.
The GAT domains of different G-type amidotransferases share significant sequence similarities. There are a number of residues within this domain that are absolutely conserved in all family members and across species. Among these invariant residues is a cysteine in a consensus sequence: PXXG(I/V)C(L/Y)G(H/M)Q, where X represents variable hydrophobic residues. This cysteine residue is thought to be important in catalysis for two prokaryotic amidotransferases, carbamyl phosphate synthetase and anthranilate synthetase ( (1) and references therein).
GMP synthetases from both prokaryotic and
eukaryotic sources contain a conserved cysteine within the GAT domain.
In human, this cysteine is at position 104 of the predicted
sequence(6) . The role for the conserved cysteine has not yet
been clearly defined for GMP synthetase from any source. For the Escherichia coli enzyme, iodoacetamide inhibits the enzyme
activity by cysteine modification(7) . However, the bulk of the
iodoacetamide alkylation was not at the conserved cysteine
(Cys) but rather a cysteine residue (Cys
)
not known to be catalytically important(8) . The sequence
surrounding Cys
has no similarity with other GMP
synthetases or any amidotransferases. It is unknown whether the
Cys
is important for the function of the GAT domain.
GMP synthetase is particularly important as a potential target for
anticancer and immunosuppressive
therapies(6, 9, 10) . An understanding of the
active site topography of a target enzyme from human is crucial for
designing potent and selective drug candidates. Recently, human GMP
synthetase has been purified and characterized(5, 6) .
Here, we report on the glutamine binding and hydrolyzing functions of
this enzyme. We show that the glutamine hydrolysis can be uncoupled
from GMP synthesis, thus distinguishing the GAT and synthetase
functions of the enzyme. An affinity probe specific for the glutamine
site was used to label an active site residue. Biochemical and physical
evidence are provided that identify the conserved cysteine
(Cys) as an active site residue essential for the GAT
function of human GMP synthetase.
Figure 1: Structure of acivicin.
On-line
HPLC-mass spectrometry was carried out using a microsyringe pump (ABI
model 140B; Applied Biosystems) interfaced to a triple stage quadrupole
mass spectrometer (TSQ700; Finnigan-MAT, San Jose, CA) equipped with an
electrospray ionization source. Separations were carried out on a Vydac
C4 column (2.1 150 mm) using a linear gradient consisting of
solvents A (0.1% trifluoroacetic acid in water) and B (0.1%
trifluoroacetic acid in 80:20 acetonitrile:water) as follows:
0-10 min, 2% B; 10-140 min, linear gradient to 100% B. The
flow rate was 100 µl/min. The column effluent was split 50:1, and 2
µl/min was directed to the mass spectrometer. The major portion of
the column effluent was directed to a fraction collector, and peptides
in each fraction were subsequently analyzed by tandem mass spectrometry
and by Edman microsequencing. Tandem mass spectrometry was performed by
using collision-assisted dissociation of the multiply charged ions,
using argon as a collision gas at pressures of 2-3 millitorr and
energies (E
) of -15 to -30 eV. Edman
microsequencing was carried out using standard methods on a ABI model
421 sequencer (Applied Biosystems). These data allowed the assignment
of all of the major components observed in the tryptic digest of GMP
synthetase and accounted for approximately 96% of the predicted
sequence of the enzyme.
Figure 2:
Inhibition of GMP synthetase by acivicin.
GMP synthetase was preincubated with acivicin at 40 °C for 10 min
in the presence of standard concentrations of Mg,
ATP, and XMP. Following the preincubation, an aliquot (3 µl) of the
preincubation mixture was withdrawn, and the GMP synthetase activity
was measured in a separate activity assay (30 µl final) as
described under ``Experimental Procedures.'' The formation of
either [
C]glutamic acid (
),
[
C]GMP (
), or [
C]AMP
(
) was measured. Initial velocity values were normalized to the
values of the ``no inhibitor'' samples to give fractional
velocity. The IC
values were calculated by non-linear
regression analysis using Systat software.
Two other
irreversible inactivators of glutamine requiring enzymes were also
tested, and they are DON and azaserine ( (13) and references
therein). GMP synthetase was preincubated as described above with each
compound prior to enzyme activity assay. The IC values are
0.4 and 40 mM for DON and azaserine, respectively (data not
shown). These latter compounds are much less potent then acivicin, and
acivicin was selected to be characterized further.
Figure 3:
Time dependence and substrate
requirements of acivicin inhibition. GMP synthetase (0.4
µM) was preincubated at 40 °C with 5 µM acivicin either alone () or with the following additions:
Mg
, ATP, XMP, and glutamine (
);
Mg
, pyrophosphate, and XMP (
);
Mg
, ATP, and XMP (
). For a control, samples
were preincubated without acivicin but with Mg
, ATP,
XMP, and glutamine (
). The substrates were at standard
concentrations, and pyrophosphate was 2 mM. After each
designated time interval, enzyme activity was assayed by
[
C]GMP formation as described in the Fig. 2legend. Data were normalized to the sample with no
preincubation and no inhibitor.
Acivicin inhibits the glutaminase activity and also inhibits the synthetase function when the only nitrogen source is glutamine. Interestingly, the effect of acivicin is dramatically different when glutamine is replaced by ammonium chloride. Whereas GMP formation in the presence of gluatmine can be completely inhibited, the reaction in the presence of ammonia is not affected at all by acivicin (Fig. 4). Similar to GMP formation, ATP hydrolysis in the presence of ammonia is also not inhibited by acivicin (data not shown). These data show that acivicin inhibition interferes only with the glutaminase activity in releasing the amino group. As long as a source of the amino group is available, such as in the presence of ammonia, the synthetase activity of GMP synthetase is not affected by acivicin. But when the amino group release by glutamine hydrolysis is blocked by acivicin, the synthetase activity is also prevented. These results clearly distinguish the two domain functions. Furthermore, they also demonstrate that the two are well coordinated.
Figure 4:
Differential effects of acivicin on
glutamine- and ammonia-dependent GMP formation and ATP hydrolysis. GMP
synthetase was inactivated by incubating the enzyme (40 µg, 0.5
µM) with acivicin (50 µM) at 40 °C for 30
min in the presence of standard concentrations of Mg,
ATP, and XMP. The unbound small molecules were removed from the protein
by gel filtration chromatography using NICK columns. Enzyme activity
was assayed immediately using either 5 mM glutamine (
)
or 500 mM ammonium chloride (
) as the amino donor. A
control (active) sample was prepared in parallel except that acivicin
was omitted. Enzyme activity was assayed as above with either glutamine
(
) or ammonium chloride (
). The formation of
[
C]GMP from [
C]XMP was
measured. Protein concentrations were determined by Bradford analysis
using bovine serum albumin as the standard.
Acivicin inhibition of GMP synthetase is not readily reversible. Enzyme activity is not recovered by dilution or by gel filtration chromatography. These data are consistent with acivicin covalently modifying GMP synthetase at the glutamine binding site.
K is the dissociation constant of the
non-covalent enzyme-inhibitor complex, k
is
the rate constant of the inactivation, [I] is inhibitor
concentration, and t is reaction time.
For the inhibition
of GMP synthetase by acivicin, pseudo-first order kinetics is observed
for the first 70-80% inactivation (Fig. 5a). From
these data, the half-time of inactivation () can be determined.
Kinetically,
= ln 2/k
,
where k
represents the first order rate
constant. However, an examination of k
as a
function of [I] reveals that within the acivicin
concentrations that were tested, saturation kinetics was not observed (Fig. 5b). Since k
= k
* [I]/(K
+ [I]), and k
versus [I] shows a linear relationship, it is likely that K
is much larger than 90 µM, the
highest [I] tested. This estimate is somewhat consistent with
the result described above that without preincubation, acivicin
inhibits GMP synthetase with an IC
of 178
µM. Based on this assumption, under the conditions tested, can be simplified as follows,
Figure 5:
Kinetics of acivicin inactivation. a, GMP synthetase was preincubated with various concentrations
of acivicin in the presence of standard concentrations of
Mg, ATP, and XMP. The following acivicin
concentrations were used: 0 µM (
), 2.5 µM (
), 10 µM (
), and 90 µM (
). After specified time intervals, aliquots were
withdrawn, and enzyme activity was determined by
[
C]GMP formation as described in the legends of Fig. 2. During the activity assay, the concentration of
glutamine was increased to 10 mM to counteract the residual
acivicin. Under these conditions, residual acivicin (up to 10
µM) causes less than 5% inhibition of enzyme activity. b,
values were determined from the data shown above as
well as at the additional acivicin concentrations, k
= ln 2/
.
where the term k/K
represents a second order rate constant. For the inactivation of
GMP synthetase by acivicin, this value is estimated to be 31.9 ±
1.3 min
mM
.
Figure 6:
Stoichiometry of acivicin inactivation.
GMP synthetase (4 µM) was preincubated with acivicin
(0-4 µM) for 60 min in the presence of standard
concentrations of Mg, ATP, and XMP. Following the
preincubation, enzyme activity was determined immediately by the
spectrophotometric coupled assay as described under ``Experimental
Procedures.'' During the activity assay, the contents of the
preincubation mixture were 100 times less
concentrated.
To determine the site of covalent modification, inactivated enzyme (acivicin treated) and active control (acivicin omitted) were prepared in parallel. Both samples were treated with dithiothreitol to reduce the cysteine residues, alkylated with iodoacetamide to modify all free cysteine residues, and then digested with trypsin. The tryptic peptides were separated by HPLC and analyzed by mass spectrometry. More than 96% of the predicted protein sequences were identified(11) . According to the observed stiochiometry of inactivation, a single site of modification was expected in the GMP synthetase sequence. A comparison of the tryptic digestion patterns reveals that the control and the inhibited samples were essentially identical except for one specific peptide, designated as T9 in the control (Fig. 7). Acivicin inhibition leads to the disappearance of the T9 peptide and the concomitant appearance of a T9a peptide. Based on molecular weight analysis and Edman microsequencing, both T9 and T9a correspond to residues 71 to 112, which reside within the predicted GAT domain of human GMP synthetase. This stretch of sequence contains a highly conserved region of the GAT domain including the invariant cysteine residue at position 104, which is the only cysteine residue in this sequence.
Figure 7: HPLC elution profile of tryptic peptides. GMP synthetase (40 µg at 3.3 µM) was treated with acivicin (6.6 µM) and applied to a NICK column equilibrated in 0.1 M ammonium bicarbonate as described under ``Experimental Procedures.'' A control sample was treated in parallel except that acivicin was omitted during the initial incubation. The treated sample was found to be 97% inhibited. Both samples were reduced, alkylated, and digested with trypsin; the tryptic peptides were separated and analyzed by on-line HPLC-mass spectrometry as described (see ``Experimental Procedures''). The digestion profile of the control sample is shown in the toppanel, and the acivicin-treated sample is shown in the bottompanel.
The observed molecular weight of
T9 is 4574.1 ± 1.0, and that of T9a is 4659.0 ± 1.5. The
former is in good agreement with the predicted mass (M = 4,575) of a peptide spanning residues 71-112 that
contains a single cysteine that has undergone modification by
iodoacetamide. The difference in the observed molecular weights between
T9 and T9a corresponds exactly to the difference in mass between
iodoacetamide and acivicin, indicating that in T9a, the iodoacetamide
modification is replaced by acivicin. The observed molecular weights of
T9a agrees with the predicted weight of residues 71-112 labeled
with acivicin (M
= 4,660).
Edman
microsequencing of both T9 and T9a confirmed the identities of residues
71 through 90. Since this subsequence contains a single cysteine
residue and the mass difference between the two peptides corresponds to
substitution of an acetamide by an acivicin moiety, these data strongly
suggest that the site of acivicin modification is Cys. Fig. 8shows the proposed structures of T9 and T9a, where
Cys
in T9 is modified by iodoacetamide and that in T9a is
modified by acivicin.
Figure 8: Proposed structures of T9 and T9a. Peptides T9 and T9a described in Fig. 7were analyzed by electrospray ionization mass spectrometry. The sequence of the first 20 residues for both peptides was confirmed by Edman microsequencing.
The appearance of T9a is well correlated with
the disappearance of T9. More importantly, it is also well correlated
with the time dependence of GMP synthetase inactivation (Fig. 9). These results show that the modification of
Cys is the cause of the inhibition of GMP synthetase by
acivicin.
Figure 9:
Correlation of Cys labeling
and inhibition of enzyme activity. GMP synthetase was incubated with
acivicin as described in the legend of Fig. 7. After specific
time intervals, samples were quenched in 50 mM EDTA and
immediately applied to NICK columns for the removal of unbound
inhibitor molecules. A control sample was incubated as above for 30 min
except in the absence of acivicin and then processed with the identical
procedures. Enzyme activity and protein concentration for all samples
were assayed immediately after the NICK elution. The amount of T9 or
T9a in each sample was determined by integrating the corresponding peak
area in HPLC profiles of tryptic digests. The amounts of T9 and T9a and
enzyme activity were corrected for the protein content in each sample.
The data were normalized to those of the control
sample.
The GAT domain of human GMP synthetase has been characterized and probed with glutamine affinity analogs. Since acivicin is the most potent of the analogs tested, it was selected as a tool to dissect the enzymatic activity of GMP synthetase. Through the action of acivicin, the synthetase and glutaminase functions of GMP synthetase are clearly distinguished since ATP hydrolysis and GMP formation can take place in the absence of glutamine hydrolysis. Acivicin fits the classical definition of a syncatalytic modifier, where it is an affinity substrate analog whose action is facilitated by substrate turnover (18) .
Enzyme inactivation by acivicin is complete and
stoichiometric. Our data strongly suggest that the site of acivicin
modification is Cys, the conserved cysteine in GAT domain
of the human sequence. This is the only modification detected, and it
correlates well with the inactivation of enzyme activity (Fig. 9). These results demonstrate the catalytic importance of
the conserved cysteine residue and are consistent with Cys
being essential for the GAT function of human GMP synthetase.
Modification of Cys
impairs the catalytic ability to
release the amino group from glutamine but without any interference
with the amination of XMP. Acivicin inactivation appears to be highly
selective toward the glutaminase function of GMP synthetase while
leaving the synthetase function unaltered (Fig. 4). These
results are evidence for not only a functional but also a physical
distinction between the GAT and the synthetase domains of GMP
synthetase, since the addition of a bulk selectively abolishes the
former without affecting the latter.
An interesting feature of
acivicin inhibition is its robust requirement for Mg,
ATP (or pyrophosphate), and XMP. If any one of these ligands is
missing, acivicin is rather inactive toward the enzyme. This could be
an indication that Cys
is dormant in a naive enzyme not
undergoing catalysis. However, in the presence of substrates, the
sulfhydryl group of Cys
becomes a highly reactive
nucleophile. In the case of acivicin modification, the enzyme cysteine
sulfhydryl group attacks carbon 3 in the isoxazole ring of acivicin and
displaces chloride. ``Activation'' of Cys
is
likely to involve a change in enzyme conformation. It is intriguing
that the same substrate requirement for acivicin inactivation is also
observed for glutamine hydrolysis. These results suggest that the
reaction toward acivicin and glutamine may require the same enzyme
conformation, which can be induced by the occupation of the
Mg
and XMP binding sites and the pyrophosphate
portion of the ATP binding site.
There are likely to be common
elements in the catalytic site that are involved in both acivicin
inhibition and glutamine hydrolysis. Cys could be such an
element. Previous studies on other amidotransferases, CTP synthetase (19) and formylglycinamide ribonucleotide
amidotransferase(20) , have demonstrated the formation of a
glutamyl-enzyme complex during catalysis where the glutamyl group is
covalently bound via a thioester linkage. A catalytic cysteine is
thought to serve as a docking site for the glutamyl group during the
hydrolysis of glutamine to release the amino group. Although there is
no direct evidence that the conserved cysteine in any amidotransferase
is the attachment site for the glutamyl group, Cys
in the
human GMP synthetase is a reasonable candidate. Since it appears that
Cys
becomes highly reactive upon the binding of
substrates, it is possible that the Cys
sulfhydryl
participates in the glutamine hydrolysis as a nucleophile releasing the
amino group, resulting in the formation of a
-glutamyl
thioester-enzyme complex.
Two functional domains of GMP synthetase
are responsible for catalyzing three reactions. The GAT domain is
involved in hydrolyzing glutamine and releasing the amino group. The
synthetase domain is involved in utilizing ATP hydrolysis to drive the
amination of XMP. The involvement of ATP was convincingly shown to be
linked to the formation of an adenyl-XMP intermediate during
catalysis(21, 22) . The displacement of the adenyl
group at the C2 position of XMP by an amino group releases AMP and
subsequently forms GMP. Presumably, the source of the amino group can
be either glutamine or ammonia. Our data show that acivicin inhibits
all three functions of GMP synthetase, namely glutamine hydrolysis, ATP
hydrolysis, and GMP formation when glutamine is the only amino source (Fig. 4). However, when ammonia replaces glutamine as the amino
source, neither ATP hydrolysis or GMP formation is affected by acivicin (Fig. 4). Since the formation of AMP is linked to the formation
of the adenyl-XMP intermediate, these results imply that adenyl-XMP
will form regardless of whether the glutaminase is functional.
Conversely, glutamine hydrolysis will occur regardless of whether the
adenyl-XMP intermediate can be formed, as long as certain pockets of
the nucleotide binding sites are occupied. The binding of
Mg, pyrophosphate, and XMP allows Cys
to be involved in catalysis in the absence of adenyl-XMP
formation. Since pyrophosphate is normally not present in cells but is
produced from ATP hydrolysis during catalysis, our data suggest that
the formation of adenyl-XMP may trigger glutamine hydrolysis.
In summary, our data show that although the two domains of GMP synthetase are distinct in functional characteristics, the activities of the two domains are well coordinated. Acivicin is a useful tool for identifying an essential active site residue and for elucidating the catalytic mechanism of GMP synthetase.