(Received for publication, November 17, 1994; and in revised form, May 18, 1995)
From the
Nitrogen regulation of transcription in Escherichia coli requires sensation of the intracellular nitrogen status and
control of the dephosphorylation of the transcriptional activator
NRI Glutamine synthetase is the most important enzyme of nitrogen
assimilation in Escherichia coli and related bacteria;
consequently this enzyme is highly regulated (reviewed in (1, 2, 3, 4, 5, 6, 7) ).
One mechanism of regulation is the concerted feedback inhibition of the
enzyme by small molecules (alanine, glycine, histidine, tryptophan,
glucosamine 6-P, CTP, AMP, and carbamyl phosphate). The enzyme is also
subjected to reversible covalent modification (adenylylation) that
regulates its activity, with the adenylylated form being much less
active. Finally, the biosynthesis of glutamine synthetase is regulated
with regard to the intracellular nitrogen status. These overlapping
controls ensure that the cell has precisely the proper level of
glutamine synthetase activity to maximize growth in a given environment
as well as the capacity to rapidly increase or decrease this activity
in response to environmental change. The regulation of the
reversible adenylylation of glutamine synthetase and the regulation of
the biosynthesis of glutamine synthetase use a common sensing
mechanism(8, 9, 10, 11, 12, 13, 14) ,
which is the subject of this paper. The key sensory components are a
signal transducing bifunctional uridylyltransferase/uridylyl-removing
enzyme (UTase/UR) The PII protein plays a key role in the regulation of nitrogen
assimilation in E. coli by controlling the activity of two
bifunctional signal-transducing enzymes: the adenylyltransferase
(ATase) responsible for the reversible adenylylation of glutamine
synthetase and the kinase/phosphatase (NRII or NtrB) responsible for
the phosphorylation and dephosphorylation of the enhancer-binding
transcription factor (NRI or NtrC) necessary for activation of the Ntr
regulon(8, 12, 16, 18, 21, 22, 23) .
The reversible metabolic transformation of PII by the UTase/UR protein
regulates the effect of PII on the ATase and
NRII(8, 12, 17, 21) . For example,
the adenylylation (inactivation) of glutamine synthetase by the ATase
is greatly stimulated by PII, whereas the deadenylylation (activation)
of glutamine synthetase is greatly stimulated by PII-UMP. The
activation of transcription of the glnA gene, encoding
glutamine synthetase, in response to nitrogen starvation requires the
phosphorylated form of the transcription factor NRI (NtrC) ((22) , reviewed in (4, 5, 6, 7) ). The nitrogen
regulation of transcription of glnA reflects the control of
the extent of NRI phosphorylation; this control is obtained by the
regulation of NRI In the current
work, we used purified components to examine the binding of small
molecules to PII and the effect of small molecules on the ability of
PII to elicit the regulated phosphatase activity, the ability of PII to
bind to immobilized NRII, and the ability of PII to become uridylylated
by the UTase/UR protein. Our results indicate that ATP and
2-ketoglutarate bind to PII with high affinity, and glutamate binds to
PII with lower affinity. These small molecules stimulate the regulated
phosphatase activity, the uridylylation of PII by the UTase/UR, and the
binding of PII to NRII. Also, we show that PII can bind to NRII in the
absence of NRI
Figure 4:
Binding of 2-ketoglutarate and ATP to PII.
The binding curves represent the fit of raw binding data (data points
shown) by nonlinear regression analysis, using the Enzfit program.
Scatchard transformations of the fit data are shown as insets. Panel A, binding of 2-ketoglutarate to PII. Binding was
determined at 1.33 µM PII and 0.1-50 µM [
Figure 5:
Binding competition by unlabeled ligands
and ligand analogs. Panel A, competition of unlabeled
2-ketoglutarate, 3-ketoglutarate, oxaloacetate, and glutamate with
labeled 2-ketoglutarate for binding to PII. PII was present at 4
µM, ATP was 2 mM, and
[
To measure the deuridylylation of
PII-UMP, PII-UMP labeled with
The raw corrected data for the uridylyl-removing activity assay are
shown as the data points in Fig. 8. The curves were also
obtained by fitting these data as single exponential decay, using the
Enzfit program. The secondary plot was fit with an offset representing
the uridylyl-removing activity observed in the absence of glutamine.
Rates were determined from the fit curves as cpm released from
PII-[
Figure 8:
Dependence of the uridylyl-removing
activity on the concentration of the activator, glutamine. Panel
A, uridylyl-removing assays were performed at 30 °C using
purified PII-UMP (
In order to study
the activation or inhibition of the NRI
Figure 1:
Effect of small
molecule effectors on the regulated phosphatase activity. NRI
During the course of our studies, we were informed by Dr. Boris
Magasanik that he and Dr. Jiu Liu had observed that glutamate at much
higher concentrations similarly stimulated the regulated phosphatase
activity, and that 2-ketoglutarate at 15 mM did not stimulate
the regulated phosphatase activity(36) . Using our assay
methods, higher concentrations of 2-ketoglutarate did not stimulate the
phosphatase activity (Fig. 1A). In the absence of PII,
2-ketoglutarate at 10 mM resulted in a reduction in the
steady-state level of NRI We
also checked the effect of glutamine and the following compounds:
glutarate, 3-ketoglutarate, oxaloacetate, aspartate, asparagine,
succinate, tartrate, pyruvate, 3-ketobutyrate, and 2-ketopentanoate. Of
these, glutamine was only weakly able to stimulate the phosphatase
activity (Fig. 1C). We cannot exclude the possibility
that this glutamine effect may be due to glutamate or 2-ketoglutarate
contamination of the glutamine preparation. (The possibility that the
glutamate effect is due to 2-ketoglutarate contamination of the
glutamate was eliminated by Liu and Magasanik(36) .) Pyruvate,
3-ketoglutarate, and oxaloacetate had a discernible effect on the
dephosphorylation of NRI
Figure 2:
Dependence of uridylyltransferase activity
on the concentration of the activator, 2-ketoglutarate. Panel A depicts the results of uridylyltransferase assays. The data points
are the raw data, corrected for background (``Experimental
Procedures''). The curves result from fitting of the data
as described under ``Experimental Procedures.'' Panel B depicts the dependence of the calculated initial rates on the
concentration of the activator. Experimental conditions: 66 µM PII, 0.066 µM UTase/UR, 500 µM UTP, 100
µM ATP, 100 mM Tris-Cl, pH 7.5, 100 mM KCl, 10 mM MgCl
The uridylylation of PII by the UTase/UR protein
could also be activated by 3-ketoglutarate and by oxaloacetate, but
these compounds were about 100-fold less effective than was
2-ketoglutarate (data not shown). Glutamate was only able to activate
the uridylylation of PII when at very high concentration (Fig. 3, Table 1). As shown, glutamate is approximately
2.5-5.0
Figure 3:
Glutamate weakly activates the
uridylyltransferase activity and does not affect the activation of this
activity by 2-ketoglutarate. Panels A and B depict
uridylylation assays. Reactions were performed as described under
``Experimental Procedures,'' assays were initiated by
addition of [
We also measured the binding of ATP to
PII in the presence and absence of 2-ketoglutarate (Fig. 4B, Table 2). ATP bound tightly to PII
(apparent K Competition experiments were also performed as an independent means
of studying the binding of various small molecules to PII in the
presence of 2 mM ATP. As shown in Fig. 5A,
unlabeled 2-ketoglutarate could effectively compete with labeled
2-ketoglutarate, while unlabeled 3-ketoglutarate, oxaloacetate, and
glutamate were much less effective competitors. The ability of
unlabeled ATP to compete with the labeled ATP for PII was also assessed (Fig. 5B). Unlabeled ATP was able to effectively
compete with the labeled ATP for PII binding, but unlabeled ADP was
100-fold less effective as competitor.
In order to detect the
binding of PII to NRII, PII was permitted to interact with MBP-NRII
under various conditions, the reaction mixtures were added to amylose
resin to immobilize the MBP-NRII, the resin was extensively washed to
remove unbound material, and the bound proteins were then eluted with
maltose (``Experimental Procedures''). As shown in Fig. 6A, the retention of PII on the immobilized NRII
required the simultaneous presence of 2-ketoglutarate,
Mg
Figure 6:
Retention of PII on immobilized NRII. PII
(25 µM) was incubated with MBP-NRII (15 µM),
mutant NRII protein fused to MBP (15 µM) or MBP (15
µM), as indicated, in 50 mM Tris-HCl, pH 7.5, 100
mM KCl, in the presence of small molecule effectors as
indicated. UTase/UR protein (15 µM) or soluble NRII were
present as indicated. The reaction mixtures were subsequently mixed
with amylose resin, washed with buffers containing the small molecule
effectors present in the initial reaction mixture, and finally eluted
with 10 mM maltose, as described under ``Experimental
Procedures.'' The eluates were analyzed for protein content by
SDS-15% polyacrylamide gel electrophoresis and Coomassie Brilliant Blue
R-250 staining. Small molecule effectors, when present, were at the
following concentrations: MgCl
To further investigate the specificity of
binding in these experiments, we examined the effect of using MBP
(lacking any NRII sequences) and two different fusion proteins
containing MBP: MBP-cNRII, which consists of MBP fused to codon 110 of
NRII, and MBP-NRII-H139N, which contains MBP fused to the indicated
point mutant version of NRII. Previous studies have indicated that
MBP-cNRII retains the NRII autokinase activity but fails to display the
NRI We have previously shown that
uridylylation of PII by the UTase/UR protein eliminates the capacity of
PII to stimulate the dephosphorylation of NRI We also
examined whether the binding of PII to MBP-NRII could be competed by
wild-type NRII (lacking the MBP portion). Wild-type NRII was able to
compete with MBP-NRII for PII (Fig. 6D). In the
aggregate, these experiments suggest that 2-ketoglutarate and glutamate
stimulate the binding of PII to NRII, and that this is the mechanism by
which these compounds stimulate the phosphatase activity of NRII.
Figure 7:
Inhibition of the uridylyltransferase
activity by glutamine. Panels A-D demonstrate the results
obtained when the indicated glutamine concentrations were present at 4
different fixed 2-ketoglutarate concentrations. Panel E shows
the % inhibition of the uridylyltransferase activity as a function of
the glutamine concentration for the four experiments shown in Panels A-D. Panel F shows the 2-ketoglutarate dependence of
the uridylyltransferase activity for the experiments shown in Panels A-D. The rates were obtained from the samples lacking
glutamine in Panels A-D. Panel A, the 2-ketoglutarate
concentration was 1 µM. Glutamine concentrations were:
(
The sensory device consisting of the PII protein and the
UTase/UR enzyme is designed to permit the bacterium to respond rapidly
to subtle alterations in the intracellular nitrogen status, and to
control the activities of the ATase and NRII enzymes appropriately.
Early studies on PII and the UTase/UR identified the factors
controlling the UTase and UR activities; of these the most important
seemed to be ATP and 2-ketoglutarate, activators of the UTase activity,
and glutamine, which inhibits the UTase activity and activates the UR
activity(13, 18) . In the current report these sensory
phenomena are examined in greater detail. We demonstrated that among
the nitrogen sensory components tested, only the PII protein bound
2-ketoglutarate and ATP at micromolar concentrations. The concentration
of 2-ketoglutarate required to activate the uridylylation reaction
corresponded to the concentration of this compound required to bind the
high-affinity 2-ketoglutarate binding site(s) of PII. Thus, we
hypothesize that the sole receptor for 2-ketoglutarate (at micromolar
concentration) in our experiments is the PII protein, and that
2-ketoglutarate indirectly activates the uridylyltransferase reaction
by allosterically altering PII. Also, we demonstrated that the liganded
form of PII was better able to elicit the regulated phosphatase
activity of NRII than was unliganded PII, and that this stimulation was
most likely due to an increased capacity of liganded PII to bind to
NRII. Both 2-ketoglutarate and ATP specifically bound with high
affinity to PII and the binding of each of these ligands was greatly
stimulated by the presence of the other. The dissociation constant of
PII for 2-ketoglutarate in the presence of excess ATP was 5.62
µM (Fig. 4, Table 2). Scatchard
transformation of the binding data suggested a stoichiometry The activation of the uridylyltransferase
activity by ATP and 2-ketoglutarate at micromolar concentrations is
probably due to the requirement for the form of PII liganded with these
small molecules. In contrast, glutamine may be directly bound by the
UTase/UR. As described
earlier(13, 16, 17, 18) , glutamine
inhibited the UTase activity and stimulated the UR activity. With
regard to the UTase activity, the concentration of glutamine required
to inhibit the initial rate 50% did not vary when the concentration of
2-ketoglutarate was varied 1000-fold. Thus, 2-ketoglutarate and
glutamine are not competing for the same site. Furthermore, the
hypothesis that the UTase/UR protein is regulated by ``the ratio
of the glutamine and 2-ketoglutarate concentrations'' appears to
be excluded by our data. It is more difficult to accurately measure
the UR activity than to measure the UTase activity, since it is
difficult to provide the substrate, PII-UMP, at very high concentration
and at very high specific activity. Nevertheless, we were able to
observe that the UR activity was only stimulated by concentrations of
glutamine Our results
demonstrating that 2-ketoglutarate stimulated the dephosphorylation of
NRI Another consideration is that in
intact cells the UTase protein is well in excess of the NRII
protein(16, 31) . Indeed, the UTase activity can be
directly measured in crude extracts of wild-type cells(16) .
Thus, the action of 2-ketoglutarate and ATP might serve to make PII
bind more efficiently to both the UTase/UR and NRII. If the UTase
activity is predominant, then this increase in binding resulting from
the interaction of 2-ketoglutarate and ATP with PII could hasten the
uridylylation of PII to an extent sufficient to offset the increased
activity of the NRI What selective advantage may
result from the separation of 2-ketoglutarate and glutamine binding
functions onto two distinct proteins? One possibility is that this
arrangement permits regulation by additional factors. For example, the
PII protein appears to bind not only 2-ketoglutarate but oxaloacetate,
3-ketoglutarate, pyruvate, and glutamate as well. The assumption that
2-ketoglutarate is the physiologically relevant allosteric ligand for
PII is based in part on the fact that neither we nor others have yet
identified any compound with greater capacity to stimulate the
uridylyltransferase activity (13) . The possibility remains
that several distinct ligands contribute to the activation of PII in vivo. Another possibility, raised by
Magasanik(36) , is that there is no selective advantage to the
current arrangement, but that it reflects the modular evolution of the
sensory apparatus. For example, one could imagine that the PII protein
may have evolved first and was itself capable of nitrogen sensation by
binding small molecules. The UTase/UR protein may have then evolved at
a later time, extending the sensory capabilities to include glutamine.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
P. This dephosphorylation is catalyzed by the bifunctional
kinase/phosphatase NRII in the presence of the dissociable PII protein.
The ability of PII to stimulate the phosphatase activity of NRII is
regulated by a signal transducing uridylyltransferase/uridylyl-removing
enzyme (UTase/UR), which converts PII to PII-UMP under conditions of
nitrogen starvation; this modification prevents PII from stimulating
the dephosphorylation of NRI
P. We used purified components to
examine the binding of small molecules to PII, the effect of small
molecules on the stimulation of the NRII phosphatase activity by PII,
the retention of PII on immobilized NRII, and the regulation of the
uridylylation of PII by the UTase/UR enzyme. Our results indicate that
PII is activated upon binding ATP and either 2-ketoglutarate or
glutamate, and that the liganded form of PII binds much better to
immobilized NRII. We also demonstrate that the concentration of
glutamine required to inhibit the uridylyltransferase activity is
independent of the concentration of 2-ketoglutarate present. We
hypothesize that nitrogen sensation in E. coli involves the
separate measurement of glutamine by the UTase/UR protein and
2-ketoglutarate by the PII protein.
(
)and the PII protein (8, 9, 10, 11, 12, 13, 14, 15) .
The uridylylation of PII by the UTase activity requires 2-ketoglutarate
and ATP, and is inhibited by glutamine, while the removal of uridylyl
groups from PII-UMP requires
glutamine(13, 16, 18) . These and other data
led to the reasonable suggestion, widely accepted, that the UTase/UR
protein responds to the intracellular ratio of the concentrations of
glutamine and 2-ketoglutarate, and that this ratio is reflected in the
extent of PII modification (for examples, see Refs. 3, 7, 19, and 20).
P
dephosphorylation(16, 22, 23) . NRI catalyzes
its own phosphorylation using either of two known sources of phosphoryl
groups, the metabolic intermediate acetyl phosphate(24) , and
the phosphorylated form of the bifunctional kinase/phosphatase NRII
(NtrB, 25). NRII binds ATP and phosphorylates itself on a histidine
residue(25, 26, 32) , and these phosphoryl
groups are then the substrate for NRI autophosphorylation. The acyl
phosphate moiety in NRI
P is quite unstable, with a half-life at
neutral pH of about 4-8 min(23, 25) . This
``self-catalyzed'' dephosphorylation has been referred to as
the autophosphatase activity(23) . The rate of
dephosphorylation of NRI
P is greatly stimulated by NRII in the
presence of the unmodified form of PII and
ATP(16, 22, 23) , and this activity is
referred to as the regulated phosphatase activity(23) . Mutants
lacking either PII or NRII lack the regulated phosphatase activity and
are unable to reduce the activation of glnAp2 transcription
driven by intracellular acetyl
phosphate(8, 24, 27) . PII is not itself a
phosphatase but rather elicits this activity from
NRII(27, 28, 29, 30) . The role of
PII with respect to the phosphatase activity is entirely
regulatory(31, 33) , and the uridylylated form of PII
cannot elicit this phosphatase activity(17) .
P, and that uridylylation of PII by the UTase/UR
prevents the binding of PII to NRII. Finally, we show that the
concentration of glutamine required to inhibit the uridylyltransferase
activity is independent of the concentration of 2-ketoglutarate
present. On the basis of these data, we hypothesize that nitrogen
sensation in E. coli involves the separate measurement of
glutamine by the UTase/UR protein and 2-ketoglutarate by the PII
protein.
Purified Proteins and Small
Molecules
Preparations of NRI, NRII, MBP-NRII (a fusion protein
consisting of the E. coli maltose-binding protein
fused to the N terminus of NRII), MBP-NRII-H139N (a fusion protein
similar to MBP-NRII except containing the H139N alteration), MBP-cNRII
(a fusion protein consisting of the maltose-binding protein fused to
codons 110-349 of NRII), UTase/UR, and PII obtained previously
were used(16, 32, 33) . E. coli maltose-binding protein (MBP) was from New England Biolabs.
Protein concentrations were determined by the methods of Lowry (38) and/or by the Bradford method (binding of Coomassie
Brilliant Blue G), using a Bio-Rad protein assay kit (Bio-Rad). The
concentrations of NRI, NRII, and MBP-NRII fusion proteins are stated in
terms of the dimer, the concentration of the UTase/UR and MBP are
stated in terms of the monomer, and the concentration of PII protein is
stated in terms of the trimer. Although PII was formerly thought to be
a tetramer, more recent data indicates that it is trimeric.(
)2-Ketoglutarate, glutamine, glutamate, glutarate,
3-ketoglutarate, oxaloacetate, aspartate, asparagine, pyruvate, and
2-ketoproprionate were the most pure available, from Aldrich, Sigma, or
Calbiochem. In the case of 2-ketoglutarate, different preparations
(Sigma and Calbiochem) were tested with similar results. Each of these
compounds was prepared as a 100 or 200 mM stock in 50 mM Tris-HCl, pH 7.5. Prior to use in assays, small molecule compounds
were diluted using 200 mM Tris-HCl, pH 7.5, as the diluent.
Control experiments indicated that, with the exception of
2-ketoglutarate, addition of the compounds at the concentrations used
here did not result in an observable change in pH when the
concentration of Tris-HCl in the reaction mixtures was 50 mM.
However, at least 100 mM final concentration of Tris-HCl was
required to maintain the pH at exactly 7.5 when 15 mM 2-ketoglutarate was used. Labeled
[
C]2-ketoglutarate,
[
C]ATP, [
-
P]UTP, and
[
-
P]ATP were from ICN or DuPont NEN.
Phosphorylation of NRI
The time course of NRI
phosphorylation and dephosphorylation was measured by trichloroacetic
acid precipitation of aliquots from reaction mixtures containing 20
µM NRI, 0.2 µM NRII, PII, and small molecule
effectors as indicated, essentially as described
previously(16, 22) . Reaction conditions were 50
mM Tris-HCl, pH 7.5, 100 mM KCl, 10 mM MgCl, 25 °C, except for one experiment (not shown)
in which the buffer concentration dependence of the effects of 15
mM 2-ketoglutarate on the rate of dephosphorylation of
NRI
P was studied. In that experiment, the effect of raising the
Tris-HCl concentration to 150 mM was examined and observed to
have no effect. In another experiment, the effect of raising the
MgCl
concentration to 25 mM was examined and
observed to have no effect. The phosphorylation of NRI was initiated by
the addition of ATP (0.5 mM, containing 0.4 mCi/ml
[
-
P]ATP) to reaction mixtures preincubated
at 25 °C. At the indicated times, reaction mixtures were split and
aliquots received buffer (50 mM Tris-HCl, pH 7.5), PII
protein, or the combination of PII and small molecule effectors, as
indicated.
Binding of 2-Ketoglutarate and ATP to PII
Binding
was measured using the same reaction conditions as in the NRI
phosphorylation assays (above) and either ultrafiltration or
equilibrium dialysis methods (for overview, see (34) and (35) ). For ultrafiltration analysis, 150- or 200-µl
reaction mixtures containing the indicated small molecules, PII, and
labeled [C]2-ketoglutarate or either
[
C]- or [
-
P]ATP were
incubated at 25 °C for 40 min. Samples were withdrawn for the
measurement of the total (bound + free) ligand. The reaction
mixtures were placed into Microcon-P10 (Amicon) filtration units, and
centrifuged at 3,000 rpm until approximately half of the original
volume had passed into the receiving chamber. Aliquots were withdrawn
from the receiving chamber for measurement of the free ligand
concentration. For the equilibrium dialysis experiments, 100-µl
reaction mixtures containing PII and small molecule effectors as
indicated were placed on one side of microdialysis cells (Bel-Art
Products) and 100 µl containing twice the indicated concentration
of labeled ligands (2-ketoglutarate or ATP) in the same buffer were
placed on the other side of the membranes. The cells were incubated for
48 h at 4 °C, after which aliquots were withdrawn from both sides
for determination of the total and the free ligand concentrations.
Quantitation of the ligands was by liquid scintillation in a Beckman
LS3801 instrument using Fisher ScintiVerse scintillant. In some
experiments bovine serum albumin was used in the place of PII as a
control, or either bovine serum albumin or NRII protein were mixed with
PII as indicated, to study the effect of NRII on the binding of small
molecules to PII. Additional experiments indicated that bovine serum
albumin did not bind 2-ketoglutarate (not shown).
Binding Data Analysis
The binding curves presented
in Fig. 4and the binding constants in Table 1were
obtained by nonlinear regression analysis of the raw data, using the
Enzfit program (Elsevier-Biosoft). Scatchard plot transformation of the
data was performed using the Enzfit program and the binding curves
generated from the nonlinear regression analysis.
C]2-ketoglutarate in the presence of
either 2 mM ATP and 2 µM MBP-NRII (
, K = 0.4 ± 0.015 µM; n =
0.35 ± 0.024 mol of 2-ketoglutarate/PII monomer) or 2 mM ATP and 0.3 mg/ml bovine serum albumin (
, K = 5.62 ± 0.35 µM; n =
0.48 ± 0.012 mol of 2-ketoglutarate bound/monomer). Panel
B, binding of ATP to PII. The binding was measured at 1.33
µM PII and 0.3-50 µM [
C]ATP in the presence of 2 mM 2-ketoglutarate (
, K = 0.24 ± 0.025
µM; n = 0.55 ± 0.01 mol of ATP/PII
monomer) or in the absence of 2-ketoglutarate (
, K = 1.49 ± 0.08 µM; n =
0.42 ± 0.01 mol of ATP/PII monomer).
Nonspecific Binding
Nonspecific binding was
measured by performing binding assays in the presence of 200,000 to
50-fold excess of unlabeled ligands, depending on the labeled ligand
concentration. At the low ligand concentrations applied, the
nonspecific binding was negligible. However, at concentrations of the
ligands exceeding 5 µM, nonspecific binding to the
filtration membranes and/or the plasticware became significant and this
was subtracted by including controls containing a high amount of
competing cold ligand, and/or controls lacking PII. At all the
concentrations tested, the nonspecific binding component did not exceed
15% of the total binding.Binding Competition Studies
Competition studies
were performed using labeled ligand (2-ketoglutarate or ATP as
indicated) and various concentrations of unlabeled ligand or analogue.
The percentage bound was derived by calculation of the fraction of
ligand bound in the presence of competitor versus the fraction
bound in the absence of competitor. Nonspecific binding was not
subtracted. For the experiment with 2-ketoglutarate as the labeled
ligand (Fig. 5A), unlabeled ATP was present at 2
mM, PII was 4 µM, and labeled 2-ketoglutarate was
2 µM; 100% binding in this experiment corresponds to 1.04
µM 2-ketoglutarate (21,000 cpm). For the experiment with
ATP as the labeled ligand (Fig. 5B), unlabeled
2-ketoglutarate was present at 4 mM, PII was 1.33
µM, and labeled ATP was 0.5 µM; 100% binding
corresponds to 0.416 µM ATP (32,000 cpm).
C]2-ketoglutarate was 2.0 µM; 100%
binding corresponds to 1.04 µM 2-ketoglutarate. The
unlabeled competitors were present at the indicated concentrations:
(
) 2-ketoglutarate, (+) 3-ketoglutarate, (
)
oxaloacetate, (
) glutamate. Panel B, competition of
unlabeled ATP and ADP with labeled ATP for binding to PII. PII was
present at 1.33 µM, 2-ketoglutarate was present at 4
mM, and [
C]ATP was present at 0.5
µM; 100% binding corresponds to 0.416 µM ATP
bound. Unlabeled ATP (
) and ADP (+) were present as
indicated.
Retention of PII on Immobilized MBP-NRII
Reaction
mixtures (55 µl) containing MBP-NRII fusion protein at 15
µM (or various altered forms of MBP-NRII as indicated, or
MBP lacking NRII sequences), 100 mM Tris-HCl, pH 7.5, 100
mM KCl, and additional proteins and small molecules, as
indicated, were incubated for 15 min at 25 °C. The mixtures were
transferred into Eppendorf tubes containing 250 µl of wet amylose
resin (New England Biolabs), which was previously washed and
equilibrated at the same conditions used in the assay. The mixtures
were gently stirred on ice for 15 min, centrifuged at 5,000 rpm in a
microcentrifuge, and the supernatants were removed for analysis. The
amylose beads were washed 3 times with 1 ml of cold 100 mM Tris-HCl, pH 7.5, 100 mM KCl, containing in addition the
same low molecular weight compounds as the initial incubation mixtures,
and finally transferred to 0.2-µm Z-spin filtration units (Gelman
Scientific) as 400-µl suspensions. The beads were centrifuged to
almost complete dryness in a microcentrifuge, and the adsorbed proteins
were immediately eluted with 55 µl of 10 mM maltose in 50
mM Tris-HCl, pH 7.5, 100 mM KCl. The eluates were
analyzed by SDS-polyacrylamide gel electrophoresis on 15% SDS gels,
which were stained with Coomassie Brilliant Blue R-250. In some
experiments PII (25 µM) was uridylylated by purified
UTase/UR protein (15 µM) prior to addition of the
MBP-NRII, as indicated. In a separate experiment, the UTase/UR protein
was added after preincubation of PII with MBP-NRII, as indicated.
Controls for nonspecific binding of PII to the amylose resin were
performed in the absence of other proteins, or in the presence of the
MBP protein (lacking NRII sequences) using the protocol described
above. The competition experiments with soluble wild-type NRII protein
were performed by including NRII at the indicated concentration in the
initial reaction mixtures.Uridylylation and Deuridylylation Assays
The
uridylylation of PII was measured as described
previously(16, 17) . Briefly, PII protein and UTase/UR
were incubated at 30 °C in reaction mixtures containing 50 mM Tris-Cl, pH 7.5, 100 mM KCl, 1 mM DTT, 10 mM MgCl, 0.1 mM ATP, and 2-ketoglutarate and
glutamine as indicated. Uridylylation was initiated by the addition of
[
-
P]UTP as indicated. Samples were removed
at the indicated times onto nitrocellulose filters, which were immersed
in 5% trichloroacetic acid, washed extensively in 5% trichloroacetic
acid, dried, and counted by liquid scintillation. In some experiments,
we examined whether Tris-Cl buffer at 50 mM was sufficient to
maintain the reaction mixtures at pH 7.5, and observed that inclusion
of Tris-Cl buffer, pH 7.5, to 100 mM led to identical results.
Some experiments performed at 100 mM Tris-Cl are presented. In
other experiments we observed that the presence of
[
-
P]UTP at 0.05 or 0.5 mM instead
of 0.1 mM made no apparent difference in the dependence of the
reaction on 2-ketoglutarate.
P was purified by fast
protein liquid chromatography (Pharmacia) on a MonoQ column as
described previously(16, 17) . Deuridylylation was
measured as described previously(16) , except that the reaction
was initiated by the addition of UTase/UR protein. Briefly, PII-UMP was
incubated at 30 °C in reaction mixtures containing 1 mM MnCl
, 50 mM Tris-Cl, pH 7.5, 100 mM
KCl, 1 mM DTT, 0.1 mM ATP, and glutamine or glutamate
as indicated. Deuridylylation of PII-UMP was then initiated by the
addition of the enzyme. At the indicated times, samples were spotted
onto nitrocellulose filters and processed as described above. Previous
experiments indicated that ATP was not required for the UR activity and
had no influence on the UR activity ((16) , and data not
shown).
Data Analysis
The raw data, corrected for the
background (counts adhering to filters that were washed with the others
but had received no sample) are shown as the data points in the
figures. The uridylyltransferase assay curves were obtained by fitting
the corrected data to the equation P = Lke, where P is the product, L is the limit value, and t is time. Curve fitting
was performed by the Enzfit program with robust weighting. Initial
rates were obtained from the fit curves. Percentage inhibition of the
uridylyltransferase activity by various glutamine concentrations was
calculated as [1 - (the initial rate in the presence of
glutamine/initial rate in the absence of glutamine)]
100.
P]UMP/min.
64,000 cpm/rxn), UTase/UR (0.1 µM),
50 mM Tris-Cl, pH 7.5, 100 mM KCl, 1 mM
MnCl
, 100 µM ATP, 1 mM DTT, and
glutamine as indicated. Reactions were initiated by the addition of
enzyme. Data points are the raw data corrected for background. Curves
were fit as single exponential decay as described under
``Experimental Procedures.'' The reactions contained: (
)
0 µM glutamine, (
) 0 µM glutamine, no
enzyme control, (
) 10 µM glutamine, (
) 25
µM glutamine, (+) 60 µM glutamine,
(▾) 100 µM glutamine, (
) 250 µM glutamine, (
) 1 mM glutamine, (
) 2.5 mM glutamine, (+) 5 mM glutamine, (
) 25
mM glutamine, (
) 25 mM glutamine but no enzyme. Panel B, dependence of the initial reaction rate on the
concentration of the activator. Initial reaction rates were calculated
from the curves shown in Panel A.
The Stimulation of the Phosphatase Activity of NRII by
PII Is Regulated by the Binding of Small Molecules to
PII
Previous studies have indicated that highly purified PII and
NRII act in concert to bring about the rapid dephosphorylation of
NRIP(16, 22, 23, 33) . A
convenient assay for this phosphatase activity involves the
phosphorylation of NRI in reaction mixtures containing NRII and
[
-
P]ATP. The incorporation of label into
trichloroacetic acid precipitable material serves as a measure of the
extent of phosphorylation of the acid-stable acylphosphate moiety in
NRI. Addition of PII to the reaction mixture after a steady state level
of NRI
P has been obtained then results in the rapid
dephosphorylation of NRI
P, reflected in the loss of
trichloroacetic acid precipitable label(16, 22) . We
have already shown that under the reaction conditions employed here
(NRI at 20 µM, NRII at 0.2 µM), the
half-maximal rate of NRI
P dephosphorylation was obtained when PII
was added to
3 µM(16) .
P phosphatase activity by
small molecules, we used concentrations of PII that were limiting
(either 0.3 µM as shown or 0.5 µM). Addition
of these suboptimal concentrations of PII resulted in the limited
dephosphorylation of NRI
P as indicated by the establishment of a
new (lower) steady-state level of NRI
P soon after the addition of
PII (Fig. 1). We then measured the effect of various small
molecules on the rate and extent of dephosphorylation of NRI
P. As
shown in Fig. 1A, micromolar concentrations of
2-ketoglutarate activate the phosphatase activity in the presence of
PII. In the absence of PII, addition of 2-ketoglutarate to identical
reaction mixtures at a concentration of 50 µM or 1 mM had no discernible effect on the extent on NRI phosphorylation (Fig. 1A). In another experiment, it was observed that
addition of 2-ketoglutarate to a concentration of 5 mM also
had no discernible effect in the absence of PII (data not shown).
P was
measured as described under ``Experimental Procedures.'' NRI
was present at 20 µM and NRII was present at 0.2
µM. Reactions were started by the addition of
[
-
P]ATP (0.5 mM). After 20 min,
either 1
reaction buffer, 0.3 µM PII, or 0.3
µM PII plus the indicated low molecular weight compounds
were added. Samples were removed at the indicated times, subjected to
trichloroacetic acid precipitation, and the acid-insoluble
radioactivity was determined by liquid scintillation. Panel A,
effects of 2-ketoglutarate. The additions were: (
) buffer,
(+) PII, (
) 50 µM 2-ketoglutarate in the
absence of PII, (&cjs3415;) 1 mM 2-ketoglutarate in the
absence of PII, (
) 10 mM 2-ketoglutarate in the absence
of PII, (▾) PII + 15 mM 2-ketoglutarate, (
)
PII + 10 mM 2-ketoglutarate, (
) PII + 1
mM 2-ketoglutarate, (
) PII + 100 µM 2-ketoglutarate, (
) PII + 0.5 µM 2-ketoglutarate. Panel B, effects of glutamate. The
additions were: (
) buffer, (+) PII, (
) 50 µM glutamate in the absence of PII, (&cjs3415;) 1 mM glutamate in the absence of PII, (▾) 15 mM glutamate in the absence of PII, (
) PII + 30 mM glutamate, (
) PII + 15 mM glutamate,
(
) PII + 5 mM glutamate, (
) PII + 0.5
mM glutamate, (
) PII + 5 µM glutamate. Panel C, effects of glutamine. The additions were: (
)
buffer, (▾) 15 mM glutamine in the absence of PII,
(+) PII, (
) PII + 50 µM glutamine,
(
) PII + 0.5 mM glutamine, (
) PII + 5
mM glutamine, (
) PII + 15 mM glutamine. Panel D, comparison of the effects of 2-ketoglutarate,
3-ketoglutarate, and oxaloacetate. The additions were: (
) buffer,
(+) PII, (
) PII + 50 µM 2-ketoglutarate,
(▾) PII + 50 µM 3-ketoglutarate, (
) PII
+ 50 µM oxaloacetate, (
) PII + 0.5
µM 2-ketoglutarate, (
) PII + 0.5 µM 3-ketoglutarate, (
) PII + 0.5 µM oxaloacetate.
P (Fig. 1A). When this
effect is taken into consideration, it is clear that elevated
2-ketoglutarate concentrations (10 and 15 mM) failed to
stimulate the regulated phosphatase activity (Fig. 1A).
Glutamate stimulated the phosphatase activity, but only at high
concentrations (Fig. 1B). Glutamate, at 50
µM, 1 mM, or 15 mM had no effect in
reaction mixtures that did not contain PII (Fig. 1B).
In a separate experiment, we observed that glutamate at 30 mM also had no effect in the absence of PII (data not shown).
P. Pyruvate was about 4-fold less active
than was glutamate (data not shown). 3-Ketoglutarate and oxaloacetate
displayed an effectiveness between that of glutamate and
2-ketoglutarate, being about 100-fold less effective than
2-ketoglutarate (Fig. 1D). The possibility that the
commercial preparations of these compounds were contaminated with
2-ketoglutarate was not investigated.
Uridylylation of PII by the UTase/UR Protein Is Activated
by 2-Ketoglutarate and Weakly Activated by 3-Ketoglutarate,
Oxaloacetate, and Glutamate
Previous results have indicated that
both ATP and 2-ketoglutarate were required activators for the
uridylylation of PII by the UTase/UR enzyme, and that glutamine is a
potent inhibitor of the uridylyltransferase
activity(13, 16, 17, 18) . We
re-examined the activation of the UTase activity by 2-ketoglutarate.
The rate of uridylylation of the PII protein by the UTase/UR at various
concentrations of these two proteins was measured, in the presence of
excess ATP (0.1 mM or 0.5 mM) and various
concentrations of 2-ketoglutarate. Since the uridylyl-removing (UR)
activity of the UTase/UR protein is greatly stimulated by
Mn, while the uridylyltransferase (UTase) activity
occurs optimally in the presence of
Mg
(13, 16, 17, 18) ,
we studied the UTase activity largely in the absence of the UR activity
by inclusion of Mg
and omission of
Mn
. The data are summarized in Table 1, and the
experimental results are shown in Fig. 2for the reaction
conditions in which both substrates, PII and UTP, are present in
excess. Under all conditions examined, the rate of uridylylation of PII
was dependent on the concentration of the activator, 2-ketoglutarate,
and the half-maximal initial rate of product formation occurred at a
concentration of 2-ketoglutarate of approximately 4.0-5.0
µM.
, 1 mM DTT.
2-Ketoglutarate concentrations were: (
) 0, (
) 1
µM, (
) 5 µM, (
) 15
µM, (
) 33 µM, (+) 132
µM.
10
-fold less effective than
2-ketoglutarate in stimulating the uridylylation of PII. Also,
glutamate did not have any discernible effect on the activation of PII
uridylylation by 2-ketoglutarate. A typical experiment in which both
glutamate and 2-ketoglutarate were present is shown in Fig. 3B. In this experiment, 1 µM
2-ketoglutarate and 50 mM glutamate provided a similar
activation of the uridylyltransferase activity. When both of these
compounds were present, their effect on the activation of PII
uridylylation seemed to be additive.
-
P]UTP. Small molecule
effectors were present in the reaction mixtures as indicated. Panel
A, comparison of the activation of uridylyltransferase by
2-ketoglutarate and glutamate. The reactions contained: (
) no
small molecule effector, (+) 33 µM 2-ketoglutarate,
(
) 1 mM glutamate, (
) 2.5 mM glutamate,
(
) 10 mM glutamate, (
) 25 mM glutamate,
(
) 50 mM glutamate, (
) 100 mM glutamate.
In the same experiment, we examined the activation by 250 µM glutamate and 33 µM glutamate; these curves
overlapped that for no small molecule effector and are not shown. Panel B, glutamate has no effect on the activation of
uridylyltransferase by 2-ketoglutarate. The reactions contained:
(
) 1 µM 2-ketoglutarate, (+) 50 mM glutamate, (▾) 1 µM 2-ketoglutarate + 50
mM glutamate, (
) 5 µM 2-ketoglutarate,
(
) 5 µM 2-ketoglutarate + 50 mM glutamate, (
) 15 µM 2-ketoglutarate, (
) 15
µM 2-ketoglutarate + 50 mM glutamate,
(
) 33 µM 2-ketoglutarate, (
) 33 µM 2-ketoglutarate + 50 mM glutamate. In the same
experiment we examined uridylylation in the absence of 2-ketoglutarate
and glutamate, this curve overlapped the abscissa and is not
shown.
2-Ketoglutarate and ATP Bind to PII
In order to
determine the target for 2-ketoglutarate, we used C-labeled 2-ketoglutarate to measure directly the binding
of 2-ketoglutarate to the individual signal transduction components
(NRI, NRII, PII, and UTase/UR). Direct measurements of the binding were
performed using an ultrafiltration method and by equilibrium dialysis
(``Experimental Procedures''). These methods are limited to
the detection of constants approximately equal to the concentration of
the target protein; we probably would not have been able to detect
binding if the dissociation constant was above 25 µM. No
specific binding of 2-ketoglutarate by the UTase/UR, NRII, NRII
P,
NRI, or NRI
P could be detected (data not shown), but
2-ketoglutarate was bound with high affinity by PII (Fig. 4A). The binding of 2-ketoglutarate by PII was
strongly dependent on the presence of ATP, which also was bound by PII
(see below); in the absence of ATP, no specific binding of
2-ketoglutarate by PII could be detected (data not shown). Various
control experiments indicated that 2-ketoglutarate was not bound by
bovine serum albumin, whether ATP was present or not (data not shown).
Furthermore, neither ADP nor CTP, GTP, or UTP at 2 mM could
stimulate the binding of 2-ketoglutarate by PII (data not shown). We
could derive approximate binding constants from the data shown in Fig. 4A. In the presence of excess ATP (2 mM),
the apparent K
for 2-ketoglutarate was
5.62 ± 0.4 µM when the ultrafiltration method was
used and 6.07 ± 1.46 µM when the equilibrium
dialysis method was used. In the presence of ATP (2 mM) and
MBP-NRII (2 µM) the K
for 2-ketoglutarate was
0.4 ± 0.015 µM (Fig. 4). Thus, MBP-NRII,
which does not itself bind 2-ketoglutarate, decreases the dissociation
constant of PII for 2-ketoglutarate 15-fold. The binding data are
summarized in Table 2.
= 1.49 ± 0.078
µM) and this binding was greatly stimulated by
2-ketoglutarate at 2 mM (apparent K
= 0.24 ± 0.025 µM).
The Retention of PII on Immobilized NRII Is Increased in
the Presence of 2-Ketoglutarate or Glutamate
Since glutamate and
2-ketoglutarate stimulated the regulated phosphatase activity of NRII
+ PII, we examined the possibility that these compounds increased
the binding of PII to NRII. For these experiments, we used the MBP-NRII
fusion protein consisting of the E. coli MBP linked to
wild-type NRII(33) . Previous studies indicated that MBP-NRII
has both autokinase and regulated phosphatase activities, although the
transfer of phosphoryl groups from MBP-NRIIP to NRI is slower than
the analogous phosphoryl group transfer from NRII
P (33) .
(
)
, and ATP. 2-Ketoglutarate at 0.5 mM was
more effective in causing the retention of PII on immobilized MBP-NRII
than was 15 mM 2-ketoglutarate (Fig. 6B).
Glutamate at 15 mM slightly stimulated the retention of PII on
immobilized NRII, while glutamine at 15 mM could not
detectably increase the retention of PII on MBP-NRII (Fig. 6B).
, 5 mM; EDTA, 10
mM; ATP, 0.5 mM; 2-ketoglutarate, 0.5 mM. Panel A, requirements for retention of PII on immobilized
NRII. Lane 1, MBP-NRII and PII protein markers; lane
2, PII + MgCl
+ ATP + 2-ketoglutarate
(lacking MBP-NRII); lane 3, EDTA + ATP +
2-ketoglutarate; lane 4, MgCl
; lane 5,
MgCl
+ ATP; lane 6, MgCl
+
2-ketoglutarate; lane 7, MgCl
+ ATP +
2-ketoglutarate. Panel B, effects of small molecule effectors
on the retention of PII on immobilized NRII. The reaction mixtures
shown in lanes 2, 4, 6, and 8 contained MgCl
+ ATP; the control reactions in lanes 1, 3, 5, and 7 contained ATP + EDTA. Other additions were: lanes 1 and 2, 0.5 mM 2-ketoglutarate; lanes 3 and 4, 15 mM 2-ketoglutarate; lanes 5 and 6, 15 mM glutamate; lanes 7 and 8, 15 mM glutamine. Panel C, comparison of
MBP-NRII, MBP-cNRII, MBP, and MBP-NRII-H139N for retention of PII. All
reactions contained MgCl
and ATP, 2-ketoglutarate was
present in reactions shown in lanes 1, 3, 5, and 7,
but was absent in reactions shown in lanes 2, 4, 6, and 8.
Lanes 1 and 2, MBP-NRII; lanes 3 and 4,
MBP-cNRII; lanes 5 and 6, MBP; lanes 7 and 8, MBP-NRII-H139N. Panel D, effect of uridylylation
of PII on retention by immobilized NRII, and binding competition by
soluble NRII. All reactions contained ATP, 2-ketoglutarate, and
MgCl
. Lanes 1-3, effect of uridylylation.
PII was preincubated with the UTase/UR as indicated in the presence or
in the absence of UTP (1 mM). Lane 1, control (no
UTase/UR, no UTP); lane 2, UTase/UR but no UTP; lane
3, UTase/UR + UTP. Lanes 4-7, competition by
soluble NRII. Lane 4, 7.5 µM NRII; lane
5, 15 µM NRII; lane 6, 22.5 µM NRII; lane 7, 30 µM NRII.
P phosphatase activity in the presence of PII(33) . The
MBP-NRII-H139N protein, in which the site of NRII autophosphorylation
has been altered to a nonphosphorylatable residue, is unable to become
phosphorylated, but is a strong phosphatase in the absence of
PII(31, 33) . The phosphatase activity of this protein
is stimulated by PII(33) . As shown in Fig. 6C,
PII was retained by MBP-NRII and MBP-NRII-H139N to approximately the
same extent. The latter result indicates that the autophosphorylation
of NRII was not required for the binding of PII to NRII. PII was not
retained by MBP in the absence of NRII, and was poorly retained by
MBP-cNRII (Fig. 6C).
P by
NRII(17) . We examined the effect on the retention of PII by
immobilized MBP-NRII of incubating PII with the UTase/UR and UTP under
conditions that cause uridylylation of PII. As shown in Fig. 6D, such incubation completely prevented the
retention of PII by immobilized MBP-NRII. In contrast, incubation of
PII with the UTase/UR in the absence of UTP was without effect, i.e. a stable complex was formed (Fig. 6D). In
additional experiments, we observed that UTP when added alone was
without effect, and that 2 mM glutamine, a potent inhibitor of
the uridylyltransferase reaction, strongly antagonized the effect of
UTase/UR + UTP (data not shown). In addition, we observed that the
antagonistic effect of UTase/UR + UTP on the retention of PII by
immobilized NRII did not depend on the order of addition of the
reaction components, i.e. the same result was obtained whether
the UTase/UR + UTP were added to PII prior to the addition of
MBP-NRII or after the addition of MBP-NRII (data not shown).
Inhibition of the UTase Activity by
Glutamine
Previous results have indicated that glutamine is a
potent inhibitor of the UTase
reaction(13, 16, 17, 18) . We
measured the inhibition of this activity by various glutamine
concentrations at different fixed 2-ketoglutarate concentrations, in
reaction mixtures containing 66 µM PII, 0.066
µM UTase/UR, 0.1 mM ATP, 500 µM [-
P]UTP, and 100 mM Tris-Cl.
As above (Fig. 2), these reaction mixtures contained
Mg
and lacked Mn
, permitting the
measurement of the uridylyltransferase activity largely in the absence
of the uridylyl-removing activity. Reaction mixtures containing all
components except UTP were assembled and preincubated, and the
uridylylation of PII was initiated by addition of UTP. In Fig. 7, data from a typical series of experiments in which
2-ketoglutarate was 1 µM, 5 µM, 33
µM, and 1 mM (Panels A-D, respectively)
are shown. As expected, glutamine inhibited the formation of product.
Surprisingly, the concentration of glutamine required to provide
half-maximal inhibition of the initial rate of PII-UMP formation was
not greatly different in these experiments, despite the fact that the
2-ketoglutarate concentration was varied 1000-fold (IC
= 43-56 µM, Fig. 7E).
These results clearly indicate that glutamine and 2-ketoglutarate are
not competing for a single site, and suggest that the UTase does not
directly bind 2-ketoglutarate. The 2-ketoglutarate requirement for the
formation of PII-UMP in these experiments was examined by comparing the
results obtained in the absence of glutamine for each of the
experiments shown in Fig. 7, A-D, this analysis
indicated that the half-maximal stimulation of product formation
occurred when 2-ketoglutarate was about 4.1 µM (Fig. 7F), in agreement with the results shown in Fig. 2and Table 1.
) 0, (
) 2.5 µM, (
) 25 µM,
(
) 60 µM, (
) 125 µM, (
) 500
µM, (
) 1 mM, (+) 2.5 mM. Panel B, the 2-ketoglutarate concentration was 5
µM. Glutamine concentrations were: (
) 0, (
)
2.5 µM, (
) 25 µM, (
) 60
µM, (
) 125 µM, (
) 500
µM, (
) 1 mM, (+) 2.5 mM. Panel C, the 2-ketoglutarate concentration was 33
µM. Glutamine concentrations were: (
) 0, (▾)
2.5 µM, (
) 25 µM, (
) 60
µM, (
) 125 µM, (
) 500
µM, (
) 1 mM, (+) 2.5 mM. Panel D, the 2-ketoglutarate concentration was 1 mM.
Glutamine concentrations were: (
) 0, (
) 2.5
µM, (
) 25 µM, (
) 60
µM, (
) 125 µM, (
) 500
µM, (
) 1 mM, and (+) 2.5
mM.
Activation of the UR Activity by
Glutamine
Previous results have indicated that the UR activity
is stimulated by glutamine, requires Mn, and is
unaffected by 2-ketoglutarate and ATP (13, 16, 17, 18) . We measured the
rate of the removal of uridylyl groups from purified PII-UMP at
different concentrations of glutamine. Typical results from such a
measurement are shown in Fig. 8A. In the absence of
glutamine, or in the presence of low concentrations of glutamine, the
rate of the removal of labeled uridylyl groups from PII-UMP was so low
that accurate estimation of these rates was difficult. However, at
concentrations of glutamine above 250 µM, sufficient
activity was observed to permit accurate estimation of the initial
rate. The concentration of glutamine required to provide the
half-maximal stimulation of this rate was
1 mM (Fig. 8B). This result suggests that the UTase/UR
protein may have two distinct sites for glutamine and that the UTase
and UR activities are separately controlled. In a separate experiment,
the ability of glutamate to activate the UR activity was examined under
the same conditions; glutamate was not able to activate the
uridylyl-removing activity of the UTase/UR protein (data not shown).
0.5
molecules of ATP and 2-ketoglutarate per PII monomer (Table 2),
however, there are at least three reasons why this conclusion regarding
stoichiometry may be inaccurate. First, determination of stoichiometry
requires accurate estimation of the PII concentration. In the studies
reported here, the PII protein concentrations were determined by the
methods of Lowry (38) and Bradford(39) ; while the
results of these methods are in concordance, we have no supporting data
that either method reliably estimates PII protein concentration.
Second, accurate estimation of the nonspecific component of the binding
reactions at elevated ligand concentrations is inherently difficult.
Finally, a fraction of the purified PII may contain ligands that were
not removed during the purification procedure. We note that only in the
presence of excess NRII and ATP was the binding of 2-ketoglutarate by
PII unambiguously saturated (Fig. 4A), and in this case
Scatchard transformation of the data indicated an apparent
stoichiometry of
1 molecule of 2-ketoglutarate/PII trimer.
Clearly, further work will be required to characterize the
stoichiometry of 2-ketoglutarate and ATP binding to PII. The
stimulation of the regulated phosphatase activity by 2-ketoglutarate
was easily observable at concentrations of 2-ketoglutarate of 0.5
µM, when PII was 0.3 µM and NRII was 0.2
µM. Since the binding of PII to 2-ketoglutarate was
greatly stimulated by NRII (apparent K
of
0.4 µM when NRII was 2 µM), under these
conditions a considerable fraction of the PII could be liganded with
2-ketoglutarate and ATP.
20-fold higher than that required to inhibit the UTase
activity. Thus, we hypothesize that the UTase/UR protein may contain
two distinct sites at which glutamine binds, and that the UTase and UR
activities are separately controlled. It is of interest that at
intermediate intracellular concentrations of glutamine, our data would
predict that the UTase/UR protein is largely inactive.
P by the combination of PII and NRII (regulated phosphatase
activity) were unexpected. Since 2-ketoglutarate is anticipated to be a
signal of carbon sufficiency (nitrogen deficiency), this compound
should, if anything, prevent the destruction of the transcriptional
activator of the Ntr regulon. Instead, the dephosphorylation of
NRI
P was hastened by 2-ketoglutarate at low concentration. At
higher concentrations of 2-ketoglutarate, however, the stimulation of
the phosphatase activity was considerably diminished (Fig. 1A), while the uridylylation of PII was only
modestly diminished (Fig. 2).
This suggests that
some component of the system may contain a site at which
2-ketoglutarate binds with low affinity. This low affinity binding site
may be on PII, NRII, or NRI. The existence of a low affinity
2-ketoglutarate binding site could not be detected in our binding
assays, due to the limitations of the methods used. Since intracellular
2-ketoglutarate has been reported to fluctuate between 0.1 and 0.9
mM in glucose-limited cultures of E. coli with excess
ammonia as the nitrogen source, depending on the growth
rate(37) , occupancy of a low affinity 2-ketoglutarate site may
play an important regulatory role in vivo. If the
stoichiometry of 2-ketoglutarate and ATP in our experiments at
micromolar concentrations is 1 molecule each/PII trimer, it is possible
that at high 2-ketoglutarate concentrations binding at the two
additional possible sites in the PII trimer have a negative effect on
the interaction of PII and NRII.
P phosphatase.
We thank Jiu Liu and Boris Magasanik for communication
of unpublished results, and Boris Magasanik, Rowena G. Matthews, Chris
Harris, James Peliska, Vincent Massey, Minor J. Coon, and David P.
Ballou for helpful discussions and for reading the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.