©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Escherichia coli PII Signal Transduction Protein Is Activated upon Binding 2-Ketoglutarate and ATP (*)

(Received for publication, November 17, 1994; and in revised form, May 18, 1995)

Emmanuel S. Kamberov (§) Mariette R. Atkinson (§) Alexander J. Ninfa (¶)

From the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Nitrogen regulation of transcription in Escherichia coli requires sensation of the intracellular nitrogen status and control of the dephosphorylation of the transcriptional activator NRIP. 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 NRIP. 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.


INTRODUCTION

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)()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).

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 NRIP 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 NRIP 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 NRIP 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) .

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 NRIP, 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.


EXPERIMENTAL PROCEDURES

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 NRIP 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.


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 [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).


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 [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.

To measure the deuridylylation of PII-UMP, PII-UMP labeled with 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.

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-[P]UMP/min.


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 (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.




RESULTS

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 NRIP has been obtained then results in the rapid dephosphorylation of NRIP, 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 NRIP dephosphorylation was obtained when PII was added to 3 µM(16) .

In order to study the activation or inhibition of the NRIP 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 NRIP as indicated by the establishment of a new (lower) steady-state level of NRIP 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 NRIP. 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).


Figure 1: Effect of small molecule effectors on the regulated phosphatase activity. NRIP 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.



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 NRIP (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).

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 NRIP. 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.


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, 1 mM DTT. 2-Ketoglutarate concentrations were: () 0, () 1 µM, () 5 µM, () 15 µM, () 33 µM, (+) 132 µM.



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 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.


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 [-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, NRIIP, NRI, or NRIP 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 Kfor 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.



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 = 1.49 ± 0.078 µM) and this binding was greatly stimulated by 2-ketoglutarate at 2 mM (apparent K = 0.24 ± 0.025 µM).

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.

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 NRIIP (33) .()

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, 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).


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, 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.



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 NRIP 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).

We have previously shown that uridylylation of PII by the UTase/UR protein eliminates the capacity of PII to stimulate the dephosphorylation of NRIP 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).

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.

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.


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: () 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).


DISCUSSION

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 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.

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 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.

Our results demonstrating that 2-ketoglutarate stimulated the dephosphorylation of NRIP 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 NRIP 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.

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 NRIP phosphatase.

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM47460. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Biological Chemistry, University of Michigan Medical School, 1301 E. Catherine, Ann Arbor, MI 48109-0606. Tel.: 313-763-8065; Fax: 313-763-4581.

The abbreviations used are: UTase/UR, uridylyltransferase/uridylyl-removing enzyme; MBP, maltose-binding protein; DTT, dithiothreitol.

R. Loo-Ogorzalek, E. S. Kamberov, M. R. Atkinson, A. J. Ninfa, and P. Andrews, manuscript in preparation.

E. S. Kamberov, M. R. Atkinson, and A. J. Ninfa, unpublished data.


ACKNOWLEDGEMENTS

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.


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