Regulation of Mycobacterium smegmatis glutamine synthetase by adenylylation

Glutamine synthetase from a Gram-positive acid-fast bacterium, Mycobacterium smegmatis, was purified to homogeneity from cells grown with glycerol-bouillon medium. Electron micrographs of the enzyme revealed a dodecameric arrangement of its subunits in two superimposed hexagonal rings, similar to the structure of glutamine synthetase of Escherichia coli. Disc electrophoresis in the presence of sodium dodecyl sulfate indicated a subunit molecular weight of 56,000. The sedimentation coefficient of the native enzyme was estimated to be 19.4S by ultracentrifugation in a sucrose gradient. Like the E. coli enzyme, the glutamine synthetase from M. smegmatis is regulated by adenylylation/deadenylylation. This conclusion was based on studies of the effect of snake venom phosphodiesterase treatment on the catalytic and spectral properties of the isolated enzyme. The AMP released from the enzyme by the phosphodiesterase was identified by thin-layer chromatography. Despite the structural similarity of both enzymes, striking differences were found between the catalytic properties of M. smegmatis and E. coli glutamine synthetases. The divalent cation specificity of the M. smegmatis enzyme was not altered by adenylylation of the enzyme, and deadenylylation of the enzyme caused a significant increase in the specific activities for both biosynthetic and transfer reactions with either Mg2+ or Mn2+.     

Inactivation of Escherichia coli glutamine synthetase by thiourea trioxide

Incubation of Escherichia coli glutamine synthetase with thiourea trioxide resulted in partial inactivation of the enzyme. This reagent specifically modifies lysine residues to form homoarginine. By amino acid analysis 2.3 ± 0.3 residues of homoarginine are produced per enzyme subunit after treatment with thiourea trioxide. Previously we determined that thiourea dioxide totally inactivated glutamine synthetase and modified both lysine and histidine residues (J. Colanduoni and J. J. Villafranca (1985) J. Biol. Chem. 260, 15,042–15,050). Thiourea trioxide reacted with the same lysine residues of glutamine synthetase as thiourea dioxide. The K_m values for the thiourea trioxide modified enzyme were determined and are 210 ± 30 μm and 10 ± 1 mm for ATP and glutamate, respectively. Both values are about threefold higher than for native enzyme assayed under the same conditions. Fluorescence titrations of native and thiourea trioxide labeled enzyme showed that ATP binding was virtually unchanged by the modification while glutamate and methionine sulfoximine bound about twofold more weakly to the modified enzyme.     

Glutamine synthetase adenylylation in permeabilized cells of Escherichia coli

Following a freeze-thaw cycle, treatment of Escherichia coli with the nonionic detergent, Lubrol WX, renders the cells permeable to small molecules but not to cytosolic proteins. After such treatment, the permeabilized cell suspensions can be assayed directly by standard procedures both for intracellular levels of glutamine synthetase and the state of adenylylation (i.e. the average number, n, of adenylylated subunits/dodecameric molecule). Permeabilization of cells from cultures containing an adequate supply of glutamine as the sole nitrogen source led to complete retention of all protein components of the bicyclic cascade that regulates the interconversion of glutamine synthetase between adenylylated and unadenylylated forms; similar treatment of glutamine-starved cells leads to selective inactivation, only, of the uridylyltransferase. When suspended in buffers containing ATP and glutamine, the value of n in permeabilized cells increased to high values (n = 11), whereas in the presence of alpha-ketoglutarate, Pi, and ATP, the value of n decreased to approximately 2.0. Time-dependent changes in n that occur during incubations of permeabilized cells in buffers containing these effectors can be arrested either by sonication at 0-4 degrees C or by the addition of cetyltrimethylammonium bromide (to inactivate adenylyltransferase). It is thus evident that Lubrol-treated cells may be used to investigate the regulation of glutamine synthetase adenylylation in situ.     

Regulation of glutamine synthetase activity by phosphorylation instead of by adenylylation

o-Phosphotyrosyl glutamine synthetase (P-GS) was isolated from highly adenylated glutamine synthetase (AMP-GS) purified from Mycobacterium phlei, by treatment with micrococcal nuclease. The physical characteristics of P-GS were quite similar to those of AMP-GS except for the UV-absorption spectrum. In either Mg2+- or Mn2+-dependent biosynthetic reactions, the kinetic properties, such as optimum pH, Vmax, and apparent Km for each of three substrates of P-GS, were found to be in good agreement with those of AMP-GS. The biosynthetic activity of P-GS was markedly increased after treatment with alkaline phosphatase similarly as in the deadenylylation of AMP-GS by snake venom phosphodiesterase treatment. These results revealed that repression of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue, without recourse to adenylylation.     

Enzymic procedures for determining the average state of adenylylation of Escherichia coli glutamine synthetase.

Under physiological conditions, the activity of the glutamine synthetase in gram-negative bacteria is inversely proportional to the number of its subunits that are adenylylated [Kingdon, H. S., Shapiro, B. m., and Stadtman, E. R., (1967), Proc. Nat. Acad. Sci. U. S. A.58, 1703 – 1710]. Six different enzymic procedures have been developed for determining the average state of adenylylation, i.e., the average number of adenylylated subunits per enzyme molecule, which can vary from 0 to 12. These methods depend on measurements of the γ-glutamyltransferase activity in assay mixtures containing Mn²⁺ at a pH where adenylylated and unadenylylated subunits are equally active and also under conditions where only unadenylylated subunits are active. The methods can be used to measure the state of adenylylation of glutamine synthetase in crude extracts with an accuracy of ±7%.     

Determination of metal-metal distances in E. coli glutamine synthetase by EPR.

This paper reports the first determination of the distance between the two metal ions (per subunit) of $\text{E. coli}$ glutamine synthetase. When Mn(II) is bound at the n₁ metal ion site its EPR spectrum is diminished in intensity but not broadened as Cr(III)-ATP or Cr(III)-ADP is bound to the enzyme. A paramagnetic spin-spin interaction is responsible for this phenomenon and a metal-metal distance of ～7 Å is calculated for enzyme - Mn(II) - Cr(III)-ATP and ～6Å for enzyme - Mn(II) - Cr(III)-ADP. The metal-metal distance changes slightly when substrates or inhibitors are also bound to the enzyme demonstrating induced conformational changes in the protein at the metal ion sites.     

Reversible adenylylation of glutamine synthetase is dynamically counterbalanced during steady-state growth of Escherichia coli

Glutamine synthetase (GS) is the central enzyme for nitrogen assimilation in Escherichia coli and is subject to reversible adenylylation (inactivation) by a bifunctional GS adenylyltransferase/adenylyl-removing enzyme (ATase). In vitro, both of the opposing activities of ATase are regulated by small effectors, most notably glutamine and 2-oxoglutarate. In vivo, adenylyltransferase (AT) activity is critical for growth adaptation when cells are shifted from nitrogen-limiting to nitrogen-excess conditions and a rapid decrease of GS activity by adenylylation is needed. Here, we show that the adenylyl-removing (AR) activity of ATase is required to counterbalance its AT activity during steady-state growth under both nitrogen-excess and nitrogen-limiting conditions. This conclusion was established by studying AR⁻/AT⁺ mutants, which surprisingly displayed steady-state growth defects in nitrogen-excess conditions due to excessive GS adenylylation. Moreover, GS was abnormally adenylylated in the AR⁻ mutants even under nitrogen-limiting conditions, whereas there was little GS adenylylation in wild-type strains. Despite the importance of AR activity, we establish that AT activity is significantly regulated in vivo, mainly by the cellular glutamine concentration. There is good general agreement between quantitative estimates of AT regulation in vivo and results derived from previous in vitro studies except at very low AT activities. We propose additional mechanisms for the low AT activities in vivo. The results suggest that dynamic counterbalance by reversible covalent modification may be a general strategy for controlling the activity of enzymes such as GS, whose physiological output allows adaptation to environmental fluctuations.     

Inactivation of bacterial glutamine synthetase by ADP-ribosylation

Glutamine synthetase from Escherichia coli was inactivated by chemical modification with arginine-specific reagents (Colanduoni, J. A., and Villafranca, J. J. (1985) Biochem. Biophys. Res. Commun. 126, 412-418). E. coli glutamine synthetase was also a substrate for an erythrocyte NAD:arginine ADP-ribosyltransferase. Transfer of one ADP-ribosyl group/subunit of glutamine synthetase caused loss of both biosynthetic and gamma-glutamyltransferase activity. The ADP-ribose moiety was enzymatically removed by an erythrocyte ADP-ribosylarginine hydrolase, resulting in return of function. The site of ADP-ribosylation was arginine 172, determined by isolation of the ADP-ribosylated tryptic peptide. Arginine 172 lies in a central loop that extends into the core formed by the 12 subunits of the native enzyme. The central loop is important in anchoring subunits together to yield the spatial orientation required for catalytic activity. ADP-ribosylation may thus inactivate glutamine synthetase by disrupting the normal subunit alignment. Enzyme-catalyzed ADP-ribosylation may provide a simple, specific technique to probe the role of arginine residues in the structure and function of proteins.     

Inactivation of Bacillus subtilis glutamine synthetase by metal-catalyzed oxidation.

Instability of Bacillus subtilis glutamine synthetase in crude extracts was attributed to site-specific oxidation by a mixed-function oxidation, and not to limited proteolysis by intracellular serine proteases (ISP). The crude extract from B. subtilis KN2, which is deficient in three intracellular proteases, inactivated glutamine synthetase similarly to the wild-type strain extract. To understand the structural basis of the functional change, oxidative modification of B. subtilis glutamine synthetase was studied utilizing a model system consisting of ascorbate, oxygen, and iron salts. The inactivation reaction appeared to be first order with respect to the concentration of unmodified enzyme. The loss of catalytic activity was proportional to the weakening of subunit interactions. B. subtilis glutamine synthetase was protected from oxidative modification by either 5 mM Mn2+ or 5 mM Mn2+ plus 5 mM ATP, but not by Mg2+. The CD-spectra and electron microscopic data showed that oxidative modification induced relatively subtle changes in the dodecameric enzyme molecules, but did not denature the protein. These limited changes are consistent with a site-specific free radical mechanism occurring at the metal binding site of the enzyme. Analytical data of the inactivated enzyme showed that loss of catalytic activity occurred faster than the appearance of carbonyl groups in amino acid side chains of the protein. In B. subtilis glutamine synthetase, the catalytic activity was highly sensitive to minute deviations of conformation in the dodecameric molecules and these subtle changes in the molecules could be regarded as markers for susceptibility to proteolysis.     

Metabolite regulation of the state of adenylylation of glutamine synthetase

When glutamine synthetase is incubated in a mixture containing adenylyltrans-ferase, the regulatory protein (P_II) and several effectors, including ATP, UTP, P_i, α-ketoglutarate, glutamine, and Mg²⁺ and/or Mn²⁺, it ultimately assumes a constant state of adenylylation. The final state of adenylylation (i.e., the number of adenylylated subunits per mole of enzyme) can vary from 0 to 12 and is specified by the concentrations and ratios of the various effectors and by the extent of uridylylation of P_II (i.e., the P_IIA:P_IID ratio). Under otherwise identical conditions, increasing the concentrations of either UTP, P_i, α-ketoglutarate, Mn²⁺, or P_IID decreases the state of adenylylation finally reached, whereas increasing the concentrations of either glutamine, ATP, or Pua increases the final state of adenylylation. The final state of adenylylation is independent of the concentrations of glutamine synthetase, adenylyltransferase, and P_II (but not of the P_IIA:P_IIDratio), and also of the initial average state of adenylylation of glutamine synthetase. Various lines of evidence show that the final state of adenylylation represents a dynamic steady state in which the rates of adenylylation and deadenylylation of glutamine synthetase are equal. It is concluded that the regulation of glutamine synthetase activity by the adenylylation mechanism utilizes a significant amount of ATP energy, but this amount is less than 0.1% that utilized directly by the glutamine synthetase in the synthesis of glutamine.     

Inhibition of the adenylylation of glutamine synthetase by methionine sulfone during nitrogenase derepression

Adenylylation of glutamine synthetase was suppressed during derepression of nitrogenase synthesis in the presence of methionine sulfone and an excess of NH₄⁺. Deadenylylation of glutamine synthetase was also promoted during nitrogenase derepression under the same conditions. These results are consistent with the hypothesis that the unadenylylated form of glutamine synthetase is required for derepression of nitrogenase.     

A source of ultrasensitivity in the glutamine response of the bicyclic cascade system controlling glutamine synthetase adenylylation state and activity in Escherichia coli

Glutamine synthetase (GS) activity in Escherichia coli is regulated by reversible adenylylation, brought about by a bicyclic system comprised of uridylyltransferase/uridylyl-removing enzyme (UTase/UR), its substrate, PII, adenylyltransferase (ATase), and its substrate, GS. The modified and unmodified forms of PII produced by the upstream UTase/UR-PII cycle regulate the downstream ATase-GS cycle. A reconstituted UTase/UR-PII-ATase-GS bicyclic system has been shown to produce a highly ultrasensitive response of GS adenylylation state to the glutamine concentration, but its composite UTase/UR-PII and ATase-GS cycles displayed moderate glutamine sensitivities when examined separately. Glutamine sensitivity of the bicyclic system was significantly reduced when the trimeric PII protein was replaced by a heterotrimeric form of PII that was functionally monomeric, and coupling between the two cycles was different in systems containing wild-type or heterotrimeric PII. Thus, the trimeric nature of PII played a role in the glutamine response of the bicyclic system. We therefore examined regulation of the individual AT (adenylylation) and AR (deadenylylation) activities of ATase by PII preparations with various levels of uridylylation. AR activity was affected in a linear fashion by PII uridylylation, but partially modified wild-type PII activated the AT much less than expected based on the extent of PII modification. Partially modified wild-type PII also bound to ATase less than expected based upon the fraction of modified subunits. Our results suggest that the AT activity is only bound and activated by completely unmodified PII and that this design is largely responsible for ultrasensitivity of the bicyclic system.     

Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy.

Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H2O and D2O solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 +/- 0.5 and 4.6 +/- 0.5 water molecules coordinated to Eu3+ bound to the n1 site of unadenylylated enzyme, GS0, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 +/- 0.5 water molecules coordinated to Eu3+ at site n2 for both GS0 and GS12. Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 +/- 1.5 A. Distances between a Tb3+ ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site n1 was observed to decrease from Kd = 0.35 +/- 0.09 microM for GS0 to Kd = 0.06 +/- 0.02 microM for GS12. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 +/- 0.9 microM and Kd = 2.6 +/- 0.7 microM for GS0 and GS12, respectively. Competition experiments indicate that Mn2+ affinity at site n1 decreases as a function of increasing adenylylation state, from Kd = 0.05 +/- 0.02 microM for GS0 to Kd = 0.35 +/- 0.09 microM for GS12. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 +/- 1.3 microM for GS0 and Kd = 4.0 +/- 1.0 microM for GS12). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991, Biochemistry 30:1413-1418), supporting the hypothesis that adenylylation regulates GS by altering substrate and metal ion affinities.     

Effect of metal ions and adenylylation state on the internal thermodynamics of phosphoryl transfer in the Escherichia coli glutamine synthetase reaction.

Experiments were conducted to study the differences in catalytic behavior of various forms of Escherichia coli glutamine synthetase. The enzyme catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia via a gamma-glutamyl phosphate intermediate. The physiologically important metal ion for catalysis is Mg2+; however, Mn2+ supports in vitro activity, though at a reduced level. Additionally, the enzyme is regulated by a covalent adenylylation modification, and the metal ion specificity of the reaction depends on the adenylylation state of the enzyme. The kinetic investigations reported herein demonstrate differences in binding and catalytic behavior of the various forms of glutamine synthetase. Rapid quench kinetic experiments on the unadenylylated enzyme with either Mg2+ or Mn2+ as the activating metal revealed that product release is the rate-limiting step. However, in the case of the adenylylated enzyme, phosphoryl transfer is the rate-limiting step. The internal equilibrium constant for phosphoryl transfer is 2 and 5 for the unadenylylated enzyme with Mg2+ or Mn2+, respectively. For the Mn2(+)-activated adenylylated enzyme the internal equilibrium constant is 0.1, indicating that phosphoryl transfer is less energetically favorable for this form of the enzyme. The factors that make the unadenylylated enzyme most active with Mg2+ are discussed.