(Received for publication, November 23, 1994; and in revised form, January 19, 1995)
From the
GMP synthetase (EC 6.3.5.2) plays a key role in the de novo synthesis of guanine nucleotides. It is a potential target for immunosuppressive therapy. Recently, the human enzyme was purified to homogeneity (Hirst, M., Haliday, E., Nakamura, J., and Lou, L.(1994) J. Biol. Chem. 269, 23830-23837). We now report the characterization of this enzyme in terms of its biochemical and kinetic properties. We found that there are distinct features of the human enzyme that has not been reported for GMP synthetase from other sources. There are two variant forms of human GMP synthetase. Their catalytic properties are very similar, although their isoelectric points are different. They most likely represent posttranslational modification variants. Magnesium ion is required for enzyme activity, and the requirement is beyond levels needed for ATP chelation. Magnesium appears to be an essential activator and there may be more than one binding site. Interaction of GMP synthetase with xanthosine 5`-monophosphate (XMP), a substrate, exhibits sigmoidal kinetics with a Hill coefficient of 1.48 ± 0.07. This positive cooperativity is not due to ligand-induced oligomerization, since GMP synthetase remains a monomer in the presence of XMP and other substrates. Decoyinine, a selective inhibitor of GMP synthetase, inhibits the human enzyme reversibly with uncompetitive inhibition kinetics toward glutamine and XMP and noncompetitive kinetics toward ATP.
Proper nucleotide metabolism is an important factor for immune cell maturation and function. Inherited defects in adenosine deaminase and purine nucleotide phosphorylase result in immunodeficiency in patients (reviewed in (1) and (2) ). Selective inhibition of IMP dehydrogenase (EC 1.1.1.205) and dihydroorotate dehydrogenase (EC 1.3.99.11), which are two essential enzymes in de novo nucleotide biosynthesis, result in immunosuppression(3, 4) . These findings suggest that lymphocytes are very sensitive to abnormal levels of nucleosides and nucleotides. Accumulation of some metabolites and depletion of others disable proper lymphocyte functions. To fully understand the importance of these metabolic pathways in immune cell functions, it is necessary to closely examine the link between the immunobiology and the cell biology of nucleotide biosynthesis. Furthermore, an understanding of the enzymatic activities in the pathways is required in order to design therapies for patients when immunomodulation is desired.
GMP
synthetase is a key enzyme in the de novo synthesis of guanine
nucleotides. It is a G-type amidotransferase catalyzing the final step
of GMP synthesis in the amination of XMP ()to GMP (). The designation of ``G-type'' is based on
sequence similarities with the trpG-encoded anthranilate
synthase component II (for a review of amidotransferases, see (5) ).
Because of the importance of guanine nucleotide synthesis in immune cell functions, GMP synthetase is a potential target for immunosuppressive therapy.
Previously, the biochemical properties and reaction mechanism of the Escherichia coli GMP synthetase were characterized(6, 7, 8) . Strong evidence was provided for the formation of an adenyl-XMP intermediate during catalysis(9, 10) . Through active site labeling and sequence comparison with other G-type amidotransferases, certain key conserved residues were identified and predicted to be essential for enzyme activity(11, 12) . Molecular cloning and genetics studies provided a better understanding of the gene organization and regulation of guanine nucleotide synthesis in bacteria (for a review, see (13) ). The gua operon in E. coli is comprised of the structural genes for the two enzymes, IMP dehydrogenase and GMP synthetase, required for the biosynthesis of GMP from IMP(14) . The gua operon is controlled by the purR repressor which uses hypoxanthine and guanine as corepressors(15, 16, 17) . Unfortunately, similar knowledge of the catalytic and regulatory features of mammalian GMP synthetases is extremely limited. Information concerning the human enzyme is not available. In order to understand the critical relationship between nucleotide synthesis and immune cell functions in humans, it is important to fully examine the functional properties of this key enzyme in nucleotide metabolism.
The human GMP synthetase has been cloned recently(18) . The enzyme is encoded by a single 2.4-kilobase message and one gene. RNA hybridization experiments reveal that the level of expression is substantially higher in proliferating, transformed cells than nontransformed cells. These expression levels are consistent with previous reports that GMP synthetase activity is noticeably higher in neoplastic and regenerating tissues than in the normal counterparts (19, 20) . Furthermore, arresting the proliferation results in the dramatic down-regulation of the expression of both message and protein (18) . These findings provide a foundation toward the understanding of the expression of an enzyme important for cell growth and development as related to the cellular regulation of purine metabolism.
Recently, we reported the purification of the human GMP synthetase (18) . The present paper is the first report of the characterization of the biochemical and kinetic properties of the human enzyme. These data are fundamental for understanding the mechanisms of enzyme catalysis and inhibitor action and hence for the discovery of potent and selective inhibitors as immunosuppressive agents.
In and , v is velocity, V is maximum velocity, K
is
Michaelis constant, S is substrate concentration, n is Hill
coefficient, I is inhibitor concentration, K
is
inhibition constant for a competitive inhibitor, and K
is inhibition constant for an uncompetitive inhibitor.
[M] is the total Mg
concentration, [ATP]
is the total ATP
concentration, [MATP] is the MgATP
concentration, [M] is the free Mg
concentration, [H] is the hydrogen ion concentration, K
is the dissociation constant for
MgATP
, K
for
MgHATP
, and K
for
HATP
(24) . The values for K
K
, and K
are 1.37
10
, 2
10
, and
1.12
10
M. These values were
calculated as the reciprocal of the recommended stability constants for
MgATP
, MgHATP
and
HATP
, respectively(26) .
To investigate
the interaction of free ATP with the enzyme, initial velocity data were
analyzed by the following equations(27, 28) . These
equations describe a situation when ATP is a competitive inhibitor
toward MgATP. is expressed in terms of
free ATP and in free
Mg
.
[MgATP] is MgATP concentration,
[Mg
] is free Mg
concentration,
[ATP
] is free ATP concentration, and K
is K
of each substrate.
Figure 1: Separation of GMP synthetase into two distinct forms by anion exchange chromatography. GMP synthetase was prepared according to the purification scheme described previously (18). The data in this figure represent the final step of the purification which is anion exchange chromatography on a Mono Q HR5/5 column. A total of 0.7 unit of enzyme was applied. The elution was accomplished using a 30-ml linear gradient of sodium chloride (0-0.25 M). Fraction 1 represents the start of the gradient. a shows the GMP synthetase activity in each fraction, and b is a Coomassie Blue-stained SDS-polyacrylamide gel. Enzyme activity was determined by the spectrophotometric coupled assay as described and the total amounts of eluted activity were reported. The materials eluted in fractions 24 and 25 (0.1 M salt, 12-12.5 ml elution volume) were designated as peak I and fraction 29 (0.12 M salt, 14.5 ml elution volume) as peak II.
Figure 2: Isoelectric focusing of GMP synthetase. Peak I and peak II GMP synthetase were prepared as described in the legend of Fig. 1. The samples (5 µg each) were electrophoresed on an isoelectric focusing gel and stained with Coomassie Blue as described under ``Experimental Procedures.'' The samples were: calibration markers in lanes 1 and 4, peak I in lane 2, and peak II in lane 3. Samples that appear homogeneous in SDS gel routinely display multiple bands in isoelectric focusing gel. These bands are likely to represent protein aggregates, since the samples were electrophoresed under nondenaturing conditions. Alternatively, they may be isoelectric variants.
We considered the possibility that these two forms of enzyme may represent different populations in equilibrium. For instance, they may be at different stages of aggregation. Rechromatography of each separately or treating each individually with 0.75 M ammonium sulfate to dissemble any aggregates prior to rechromatography does not alter the elution position (not shown). Alternatively, perhaps one form represents a reaction intermediate. To test this possibility, each form was incubated with saturating amounts of substrates to allow single or multiple turnovers. Subsequent rechromatography showed that the elution profiles did not change. These results indicate that the two forms of GMP synthetase are not in equilibrium, and they are not readily interconvertible.
Both forms of GMP synthetase were characterized,
and they were found to have very similar kinetics properties. The
catalytic turnover numbers (k are 5.4
s
and 5.6 s
for peaks I and II,
respectively. The K
values toward ATP and
glutamine are 132 ± 7 µM and 406 ± 49
µM for peak I and 180 ± 12 µM and 358
± 34 µM for peak II, respectively. The interaction
of GMP synthetase with ATP and glutamine obeys Michaelis-Menton
equations. However, GMP synthetase displays sigmoidal kinetics when
interacting with XMP (Fig. 3). This phenomenon is observed
reproducibly for both forms of GMP synthetase. The half-saturation
values toward XMP are 35.6 ± 1.8 µM and 45.4
± 5.3 µM for peak I and II with the Hill
coefficients of 1.48 ± 0.07 and 1.54 ± 0.16,
respectively.
Figure 3:
Binding of XMP to GMP synthetase. The
initial velocity of peak I GMP synthetase was determined at various
concentrations of XMP. The other substrates were at saturating
concentrations as described under ``Experimental
Procedures.'' Enzyme activity was determined by
[C]GMP formation from
[
C]XMP, where the specific radioactivity was
constant at 10 mCi/mmol in all samples. The data were normalized to
maximum velocity (V
) and shown as
fractional velocity (
) in an Eadie-Hofstee plot. The lines
represent theoretical data where the Hill coefficient is 1 (
), 1.48(- - - -), or 2
(-).
Both forms of the enzyme are inhibited by a selective
GMP synthetase inhibitor decoyinine, and the modes of inhibition are
identical. Decoyinine is an uncompetitive inhibitor with respect to
both XMP and glutamine. The K values toward XMP
and glutamine are 50.4 ± 4.1 and 46.7 ± 4.6 µM for peak I, and 41.7 ± 5.3 and 43.1 ± 3.8 µM for peak II, respectively. The inhibition toward ATP is
noncompetitive. The K
and K
values are 30.4 ± 1.3 µM and 46.4 ±
6.1 µM for peak I, and 23.6 ± 3.0 µM and 29.0 ± 6.9 µM for peak II, respectively
(see Fig. 4for peak I; data not shown for peak II).
Figure 4:
Inhibition of GMP synthetase by
decoyinine. Inhibition of peak I GMP synthetase by decoyinine is
presented in double-reciprocal plots. Enzyme activity were measured as
described under ``Experimental Procedures,'' and all
substrates except for the one that was varied were at saturating
concentrations. a, ATP was the varying substrate and
decoyinine was present at 0 µM (), 50 µM (
), 100 µM (
), and 200 µM (
). b, glutamine was the varying substrate, and
decoyinine was present at 0 µM (
), 50 µM (
), 100 µM (
), and 150 µM (
). The initial velocity was normalized to V
. The fractional velocity relative to V
was fitted to the equations of Cleland
for noncompetitive inhibition (varying ATP in a) and uncompetitive
inhibition (varying glutamine in b). The lines represent the theoretical data based on the
fit.
Since the two forms of GMP synthetase are not distinguishable catalytically, and peak I is higher than peak II in both purity and availability, peak I is selected for further characterization. The remaining results represent those of peak I, which will be referred to simply as ``GMP synthetase.''
GMP synthetase converts XMP to GMP with
stoichiometric hydrolysis of ATP to AMP and inorganic pyrophosphate,
and L-glutamine to L-glutamic acid. The catalytic
rate of GMP formation is identical to those of AMP and glutamic acid
(data not shown). Ammonia can also serve as the amino group donor but
the affinity is much lower. The K of ammonia is
5.1 ± 0.6 mM. (
)The ammonia-dependent k
is 50-60% of the glutamine-dependent
activity. (
)
Figure 5:
Dependence on metal ions. GMP synthetase
activity was assayed as described under ``Experimental
Procedures'' except that concentrations of ATP and magnesium
chloride were modified as indicated. The enzyme activity is represented
by the amount of GMP formed in 10 min by 70 ng of GMP synthetase at 40
°C and pH 7.8. a, ATP was present at 2 mM (total)
with the following metal ions: Mg (
),
Mn
(
), Zn
(
), and
Ca
(
). b, total ATP concentration was
0.1 mM. The maximum rate of product formation under these
conditions is 1 nmol/10 min. Enzyme activity is represented by
-
-; the concentrations of
MgATP
(-
-
-
-) and free ATP
(- - - -) were calculated as described under
``Experimental Procedures.'' The arrow points to 95
µM MgATP
and 5 µM free
ATP. The inset represents an expansion of the lower
Mg
concentration data. c, total
Mg
concentration was 1 mM. Enzyme activity
is represented by -
-. The
concentrations of free Mg
(- - -
-), free ATP (
), and
MgATP
(
-
-
-
) were calculated
as described under ``Experimental Procedures.'' d,
initial velocity was measured in the presence of 0.049 mM (
), 0.099 mM (
), and 0.198 mM (
) MgATP
.
The data in Fig. 5c suggest
that Mg is an essential activator of GMP synthetase.
In this experiment, Mg
was lowered to 1 mM in order to decrease the ratio of free Mg
to
free ATP. Under these conditions, ATP initially causes an increase in
enzyme activity, since MgATP
is formed. As ATP
continues to increase, it causes a depletion of free Mg
and a parallel decrease in enzyme activity. In spite of the
increase in MgATP
, the enzyme activity continues to
decrease until it is completely abolished. Note that the maximum
activity does not occur at the point of maximum concentration of
MgATP
, but at a concentration lower than that, while
there is still free Mg
. These data suggest that
MgATP
alone is not sufficient for catalysis and the
presence of free Mg
is essential.
Another possible
interpretation of the data in Fig. 5c is that ATP may
be an inhibitor competing with MgATP, the true
substrate of the reaction. Thus removal of the free ATP through
Mg
chelation results in the apparent activation by
Mg
. However the present data suggest that this is
unlikely since the observed Mg
requirement is such
that enzyme activation is not correlated with the decrease of free ATP.
At low Mg
concentrations, GMP synthetase is rather
inactive even when the ratio of free ATP to MgATP
is
low. For example, at 0.4 mM free Mg
when
free ATP is 5 µM and MgATP
is 95
µM, the enzyme activity is only 10% of maximum (see arrow in Fig. 5b).
Previously, a
mathematical expression has been derived for a situation when free ATP
binds to the catalytic site where MgATP binds and
forms an abortive complex ((27) ; see and under ``Experimental Procedures''). predicts that if ATP is a competitive inhibitor toward
MgATP
, a double-reciprocal plot of initial velocity
against free Mg
concentration, as a function of
MgATP
concentration, would result in a parallel
pattern(28) . To investigate the effect of free ATP on GMP
synthetase, initial velocity was measured at several MgATP
concentrations while varying Mg
concentrations; Fig. 5d clearly shows that the initial velocity data of
GMP synthetase exhibits not a parallel, but an intersecting, pattern.
These results are inconsistent with the competitive inhibition by free
ATP as the major reason for the activation by Mg
. So
the observed inhibition by ATP is likely to be caused by ATP
diminishing the free Mg
, which is essential for GMP
synthetase activity.
Next the inhibition by products was examined (Table 1).
Inorganic pyrophosphate is the most effective product inhibitor and it
is purely competitive toward ATP. The inhibition of inorganic
pyrophosphate is not due to the depletion of MgATP caused by the chelation of Mg
. On the contrary,
AMP is a poor product inhibitor, and the inhibition is mixed type
toward ATP. Glutamic acid is virtually inactive as an inhibitor,
whereas GMP inhibits relatively effectively.
Inhibition of E. coli GMP
synthetase by psicofuranine appears to be dependent on XMP,
Mg, and pyrophosphate, and it has been described as
essentially irreversible(30) . We found the inhibition of human
GMP synthetase by decoyinine and the other adenosine analogs to be
fully reversible and not dependent on any substrate or products.
GMP synthetase is a glutamine-dependent amidotransferase that uses ATP as the driving force generating AMP and inorganic pyrophosphate. The affinity of the human enzyme toward each substrate is such that the intracellular rate of GMP formation should not be limited by ATP or glutamine, but rather be dependent on the level of XMP, since XMP is generally present at extremely low levels in cells. The human enzyme can also use ammonia in place of glutamine, although the reaction is insignificant at physiological concentrations of ammonia. As expected for a key enzyme in a metobolic pathway, GMP synthetase has very stringent requirements for substrate recognition.
Only one form of GMP synthetase in E. coli(7) or
Yoshida sarcoma ascites cells (19) has been described. However,
human GMP synthetase exists as two distinct forms that have different
isoelectric points. Since the human GMP synthetase is encoded by one
gene and one message(18) , it is unlikely that these forms
represent isozymes of distinct gene products or splice variants. More
importantly, the two forms of GMP synthetase were also observed when
the cDNA of GMP synthetase was expressed in a baculovirus system. ()The two forms do not represent different oligomeric states
of the enzyme, since they are both monomers. There is also no evidence
for the two to be reaction intermediates since they appear to be stable
and not in equilibrium. It is likely that the two forms of GMP
synthetase represent posttranslational modification variants. In spite
of their difference in net charge, the two forms of enzymes are
catalytically similar. Nothing is known about their regulatory
properties and this topic is currently being investigated.
Examination of the metal ion dependence reveals that Mg is an activator of GMP synthetase. The half-saturation value for
Mg
is close to 2 mM, regardless of whether
ATP is at 0.1 or 2 mM. The Mg
activation
appears to be essential for enzyme activity, since the complete
chelation of free Mg
by ATP results in the complete
inactivation of the enzyme (Fig. 5c). If Mg
is not essential, the activity is expected to decrease to a
certain plateau but not diminished completely. The decrease in enzyme
activity parallels the depletion of free Mg
, not the
increase of free ATP. It is unlikely that the observed requirement for
free Mg
is due to inhibition by free ATP, since the
activation of enzyme activity is not correlated with the decrease of
free ATP (Fig. 5b). Furthermore, the initial velocity
pattern in Fig. 5d suggests that any contribution of
ATP inhibition to the observed Mg
activation, if
exists at all, would be minor. Our results show that there may be more
than one Mg
binding site, although it is not clear
whether binding at one site augments the binding at subsequent site(s).
Multiple Mg
sites has not been described for GMP
synthetases from other sources.
Interaction of the enzyme with XMP
exhibits sigmoidal kinetics (Fig. 3). This phenomenon has not
been reported for any bacterial or mammalian GMP synthetase. The
apparent positive cooperativity is observed, and the Hill coefficient
remains the same when activity is measured by
[H]AMP formation,
[
C]glutamine formation, or when ATP or glutamine
is not saturating (twice the respective K
value).
Previously, it was shown that CTP synthetase, a G-type amidotransferase
involved in pyrimidine nucleotide biosynthesis, undergoes
nucleotide-induced tetramerization that leads to cooperativity in
substrate binding(31) . Unlike CTP synthetase, the sigmoidal
kinetics of GMP synthetase with XMP cannot be explained by
substrate-induced oligomerization. The presence of saturating levels of
XMP, either alone or in combination with ATP or glutamine, did not
affect the monomeric composition of GMP synthetase.
The
possible existence of multiple XMP binding sites in a monomeric GMP
synthetase is currently under investigation.
Inhibition by decoyinine exhibits uncompetitive kinetics toward XMP and glutamine and noncompetitive toward ATP (Fig. 4). These results suggest that decoyinine binding to GMP synthetase occurs after the binding of XMP and glutamine. Decoyinine may bind to the enzyme either before or after ATP binds. Previously, it was proposed for the E. coli GMP synthetase that ATP was the first substrate to bind, followed by XMP and then glutamine(10) . The current decoyinine inhibition data with the human enzyme are not entirely consistent with this proposal. According to the above order of substrate binding, if decoyinine binds after XMP and glutamine, then one would predict the inhibition to be uncompetitive toward ATP as well. We are currently pursuing the order of substrate binding for the human enzyme.
Our studies of GMP synthetase reveal some similarities between the enzyme from human and from other sources. More interestingly, we have identified some features that have not been described for any bacteria or mammalian GMP synthetases. These findings serve as a foundation to further investigate the mechanisms of enzyme catalysis and regulation. The knowledge concerning the human enzyme provides crucial information toward the search for immunosuppressive drugs.