Regulation and properties of glutamine synthetase purified from Bacillus cereus

The glutamine synthetase from Bacillus cereus IFO 3131 was purified to homogeneity. The enzyme is a dodecamer with a molecular weight of approximately 600,000, and its subunit molecular weight is 50,000. Both Mg2+ and Mn2+ activated the enzyme as to the biosynthesis of L-glutamine, but, unlike in the case of the E. coli enzyme, the Mg2+-dependent activity was stimulated by the addition of Mn2+. The highest activity was obtained when 20 mM Mg2+ and 0.5 mM Mn2+ were added to the assay mixture. For each set of optimal assay conditions, the apparent Km values for glutamate, ammonia and a divalent cation X ATP complex were 1.03, 0.34, and 0.40 mM (Mn2+: ATP = 1: 1); 14.0, 0.47, and 0.91 mM (Mg2+: ATP = 4: 1); and 9.09, 0.45, and 0.77 mM (Mg2+: Mn2+: ATP = 4: 0.2: 1), respectively. At each optimum pH, the Vmax values for these reactions were 6.1 (Mn2+-dependent), 7.4 (Mg2+-dependent), and 12.9 (Mg2+ plus Mn2+-dependent) mumoles per min per mg protein, respectively. Mg2+-dependent glutamine synthetase activity was inhibited by the addition of AMP or glutamine; however, this inhibitory effect was suppressed in the case of the Mg2+ plus Mn2+-dependent reaction. These results suggest that the activity of the B. cereus glutamine synthetase is regulated by both the intracellular concentration and the ratio of Mn2+/Mg2+ in vivo. Also in the present investigation, a potent glutamine synthetase inhibitor(s) was detected in crude extracts from B. cereus.     

Conformational changes in Mycobacterium smegmatis glutamine synthetase induced by certain divalent cations

Manganese ion, like Mg2+, has been found to produce high biosynthetic activity of the unadenylylated form of glutamine synthetase obtained from Mycobacterium smegmatis, and the activity with each of these cations was decreased by the adenylylation of the enzyme. Further, the gamma-glutamyltransferase reaction was catalyzed in the presence of either Mn2+, Mg2+, or Co2+ with both unadenylylated and adenylylated enzyme; however, each of these divalent cation-dependent activities was also decreased by one order of magnitude by adenylylation of the enzyme. From studies of UV-difference spectra, it was found that the ability of M. smegmatis glutamine synthetase to assume a number of distinctly different configurations was the result of the varied response of the enzyme to different cations. When either Mn2+, Mg2+, Ca2+, or Co2+ was added to the relaxed (divalent cation-free) enzyme at saturated concentration, each produced a similar UV-difference spectrum of the enzyme, indicating that the conformational states induced by these cations are similar with respect to the polarity of the microenvironment surrounding the tyrosyl and tryptophanyl groups of the enzyme. The binding of Cd2+, Ni2+, or Zn2+ to the relaxed enzyme each produced a different shift in the UV-absorption spectrum of the enzyme, indicating different conformational states. The kinetics of the spectral change that occurred upon addition of Mn2+, Mg2+, or Co2+ to a relaxed enzyme preparation were determined. The first-order rate constants for the decrease in relaxed enzyme with Mn2+ and Mg2+ were 0.604 min-1 and 0.399 min-1, respectively, at 25 degrees C, pH 7.4. The spectral change with Co2+ was completed within the time of mixing (less than 4 s). For these three metal ions, the total spectral change as well as the time course of the change were the same for both the unadenylylated enzyme and the partially adenylylated enzyme. However, Hill coefficients obtained from spectrophotometric titration data for both Mn2+ and Mg2+ were decreased with adenylylated enzyme to compared with unadenylylated enzyme. These results suggest that covalently bound AMP on each subunit may be involved in subunit interactions within the dodecamer. Circular dichroism measurements also indicated that the various structural changes of the M. smegmatis glutamine synthetase were produced by the binding of the divalent cations.     

林肯链霉菌谷氨酰胺合成酶的酶学性质

在分离纯化的基础上,报道了pH、温度和金属离子对林肯链霉菌(Streptomyceslincolnensis)Z-512谷氨酸胺合成酶(GS)活力的影响及GS底物专一性的研究结果.在动力学性质的研究中,发现林肯链霉菌GS在生物合成反应系统中,对底物NH_4CI的饱和曲线不遵守米氏方程.Hill作图呈两相曲线.在NH_4CI浓度低的情况下,Hill系数大于1,具有正协同效应;当NH_4CI浓度增加到一定程度时,Hill系数小于1,具有负协同效应.这说明NH_4CI不仅作为林肯链霉菌GS的底物,而且作为一种效应物调节GS的活性.林肯链霉菌GS对底物Glu及ATP的饱和曲线遵守米氏方程.在不同的激活离子存在下,GS对Glu、ATP的Km值也不同.     

Regulation and biochemical characterization of the glutamine synthetase of azotobacter vinelandii

We have investigated the regulation of the activity and synthesis of the glutamine synthetase (l-glutamate:ammonia ligase (ADP-forming), EC (6.3.1.2) of Azotobacter vinelandii. Synthesis of the enzyme was not repressed by NH+4 and/or a number of amino acids in the growth medium; however, biosynthetic activity was rapidly lost through adenylylation in response to ammonium ion. The enzyme could be prepared as a 'relaxed, divalent-cation-free form which was catalytically inactive. The 'taut', active form could be restored with 1-5 mM Mg2+, Mn2+, Ca2+ or CO2+ and taut-vs.-relaxed difference spectra unique to each divalent cation were generated. Mg2+ and CO2+ each supported biosynthetic catalysis, but with different substrate Km and Vmax values. L-Alanine, glycine and L-aspartate were the most potent of several inhibitors of the biosynthetic and the gamma-glutamyl transferase activities; only aspartate and AMP behaved differentially toward glutamine synthetase adenylylation state: the more highly adenylylated enzyme was more severely affected. Any two of alanine, glycine or AMP showed cumulative inhibition, while the inhibitory effects of groups of three effectors were not cumulative. The Co2+-supported biosynthetic activity of Al vinelandii glutamine synthetase was markedly less sensitive to inhibition my glycine and alanine and was stimulated up to 50% by 1-10 mM aspartate.     

Mg2+ is bound to glutamine synthetase extracted from bovine or ovine brain in the presence of L-methionine-S-sulfoximine phosphate

Purified glutamine synthetase from bovine or ovine brain had no tightly bound Mn2+. By extraction of bovine or ovine brain glutamine synthetase in the presence of L-Met-S-sulfoximine phosphate and ADP in metal ion-free water and 0.1 M KCl, only endogenously bound divalent cations were trapped on the enzyme. Enzyme complexes isolated by immunoprecipitation contained less than 0.05 Mn2+ and 1.5 +/- 0.2 Mg2+ per subunit. Without inactive complex formation, the enzyme immunoprecipitated from extracts contained undetectable Mn2+ (less than 0.01 eq per subunit) and 0.1-2.0 eq of Mg2+ per subunit. Direct binding measurements showed that the purified bovine brain enzyme contained two divalent cations bound at the active site of each subunit. Thus, although either Mg2+ or Mn2+ supports enzyme activity in vitro, Mg2+ rather than Mn2+ appears to be bound to brain glutamine synthetase in vivo.     

The role of specific cations in regulation of cyanobacterial glutamine synthetase

Purified glutamine synthetase from the cyanobacterium Anabaena cylindrica required a divalent cation for activity. Maximum biosynthetic activity required Mg2+ (25 mM when supplied alone). Co2+ and Mn2+ each supported up to 20% of this activity; 12 other cations tested were ineffective. At 2.5 - 10 mM Mg2+, 0.1 mM Co2+ or ethylene glycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) stimulated GS activity to maximum rates; other divalent cations (particularly Mn2+) inhibited Mg2+-dependent activity. At 5 mM Mg2+ the Kappm for NH+4 (0.05 mM) was 20-fold lower than at 25 mM Mg2+; added Co2+ did not markedly alter this low Km for NH+4; this could be physiologically important.     

Regulation of fodder yeast Candida tropicalis glutamine synthetase activity by the end products of glutamine metabolism]

Effect of different products of glutamine metabolism on the activity of glutamine synthetase in the presence of Mg2+, and Mn2+ and Co2+ as cofactors is studied. All the metabolites studied are found to inhibit the glutamine synthetase activity in the presence of any cation listed. The degree and the character of the inhibition by one or other metabolite depended in a considerable degree on the nature of the cation presented in the reaction mixture (Mg2+, Mn2+ or Co2+). The mechanism of the cumulative effect of retroinhibitors under the change of Mg2+ or Mn2+ in the reaction mixture was the same.     

北京棒状杆菌AS1.299谷氨酰胺合成酶活力调节的研究

北京棒状杆菌(Corynebacterium pekinense AS1.299)谷氨酰胺合成酶的转移酶活性依赖于Mn~(++),酶的生物合成酶活性依赖于Mg~(++),其他二价金属离子只能部分代替Mn~(++)和Mg~(++)的作用。Mn~(++)对ATP或ADP的克分子比对酶活力起调节作用。ATP、CTP,丙氨酸和甘氨酸对谷氨酰胺合成酶有较强的抑制作用;丝氨酸、谷氨酸和6-磷酸葡萄糖胺对酶活力的抑制作用分别是24,15和21％。效应物混合物对酶的作用被证明是累积性的抑制作用。     

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.     

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.     

Rat renal gamma-glutamyltransferase activity: a reaction of glutamine synthetase

The experimental observations of Welbourne (Am. J. Physiol. 226, 544-548 (1974)) led him to propose that a gamma-glutamyltransferase activity contributes significantly to basal renal ammonia production. He measured the transferase activity in vitro as gamma-glutamylhydroxamate or ammonia production from glutamine and hydroxylamine. We report that in crude homogenates of rat kidney, gamma-glutamyltransferase activity requires the presence of a divalent cation, arsenate, and ADP. These conditions are also required for the maximal expression of the gamma-glutamyltransferase reaction catalyzed by glutamine synthetase. Glutamine synthetase and gamma-glutamyltransferase activities exhibit an identical distribution during differential centrifugation. In addition, the two activities comigrate sucrose gradient velocity centrifugation, and exhibit identical heat inactivation and L-methionine sulfoximine inhibition profiles. Furthermore, both activities are found to be localized primarily in the outer stripe region of the renal medulla. Based on these observations, it is concluded that the primary gamma-glutamyltransferase activity as assayed in rat renal tissue is a partial reaction of glutamine synthetase, an enzyme which does not catalyze the production of ammonia and gamma-glutamyl peptides from glutamine.     

Low- and high-activity forms of glutamine synthetase from Rhodospirillum rubrum: sensitivity to feed-back effectors and activation of the low-activity form.

Glutamine synthetase from Rhodospirillum rubrum can be isolated in two forms, with low and high activity, respectively, depending on the concentration of combined nitrogen in the medium before harvest. The two forms have been studied with respect to their dependence on Mn2+ and Mg2+ in both the transferase and the biosynthetic assay. There is no difference in pH optimum between the forms in the biosynthetic assay. In addition the pH-optima for the two cations studied are very close, 7.4 (Mg2+) and 7.2 (Mn2+). It also shows that the activity of the low-activity form is higher than that of the high-activity form in the Mn(2+)-dependent biosynthetic assay. The two forms of Rsp. rubrum glutamine synthetase have also been studied with respect to their sensitivity towards feed-back effectors. In the transferase assay both forms are inhibited to essentially the same degree by alanine, glycine, histidine, AMP, CTP and UTP, CTP being the most effective of the nucleotides and of the amino acids alanine causes the highest inhibition. In the biosynthetic assay these effectors show different degrees of inhibition on the two different forms; the high-activity form being the most sensitive. The results are discussed in relation to properties of glutamine synthetase from Escherichia coli and other phototropic bacteria in which regulation of glutamine synthetase is known to be due to adenylylation. It is also shown that the low-activity form of Rsp. rubrum glutamine synthetase can be activated in crude extracts in a reaction that is inhibited by glutamine.     

Fluorometric studies of aza-epsilon-adenylylated glutamine synthetase from Escherichia coli

Glutamine synthetase in Escherichia coli is regulated by adenylation and deadenylation reactions. The adenylation reaction converts the divalent cation requirement of the enzyme from Mg2+ to Mn2+. Previously, the catalytic action of unadenylated glutamine synthetase was elucidated by monitoring the intrinsic tryptophan fluorescence change accompanying substrate binding. However, due to the lack of changes in the tryptophan fluorescence, a similar study could not be done with the adenylated enzyme. In this study, therefore, an extrinsic fluor is introduced into the adenylated glutamine synthetase by adenylating the enzyme with 2-aza-1,N6-ethenoadenosine triphosphate, a fluorescent analog of ATP. The modified enzyme (aza-epsilon-glutamine synthetase) exhibits catalytic and kinetic properties similar to those of the naturally adenylated enzyme. The results of fluorometric studies on this aza-epsilon-glutamine synthetase indicated that L-glutamate and ATP bind to both Mn2+ and Mg2+ forms of the enzyme in a random order, but only the Mn2+ form is capable of forming a highly reactive enzyme-bound intermediate which is a prerequisite for the reaction with NH4+ to form products. The extrinsic fluorescence changes are also used to determine the binding constants of various substrates and inhibitors of both the biosynthetic and gamma-glutamyl transfer reactions.     

Regulation and properties of the glutamine synthetase purified from Photobacterium phosphoreum

Glutamine synthetase from a marine enterobacterium, Photobacterium phosphoreum, was purified to homogeneity from cells grown in glycerol-yeast extract medium. The purified enzyme had a molecular weight of approximately 670,000 and a subunit size of 56,000, i.e. larger than that of the enzyme from E. coli. Regulation of the glutamine synthetase activity by adenylylation/deadenylylation was demonstrated on snake venom phosphodiesterase treatment. The state of adenylylation appeared to influence both the biosynthetic and gamma-glutamyltransferase activities of P. phosphoreum glutamine synthetase similar to in the case of the E. coli enzyme. The enzyme activity was controlled by adenylylation and possibly in combination with feedback inhibition by alanine, serine, and glycine, metabolites which are especially effective in inhibiting P. phosphoreum glutamine synthetase. When either Mn2+ or Mg2+ was added to the relaxed (divalent cation-free) enzyme, similar UV-difference spectra were obtained for the enzyme, indicating that the conformational states induced by these cations were also similar. The profile of these spectra varied from those published for E. coli, and three peaks were four 1 at 282.5, 288.5, and 298 nm.     

Identification of the reactive cysteinyl residue and ATP binding site in Bacillus cereus glutamine synthetase by chemical modification

Bacillus cereus glutamine synthetase was modified by reaction with a fluorescent SH reagent, N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS), or an ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The locations of the specific binding sites of these reagents were identified. IAEDANS inactivated Mg2(+)-dependent activity and activated Mn2(+)-dependent activity. FSBA inactivated only Mn2(+)-dependent activity. Mg2+ plus Mn2(+)-dependent activity was inactivated by IAEDANS or FSBA. Amino acid sequence analysis of the single AEDANS-labeled proteolytic fragment showed the cysteinyl residue at position 306 to be the site of modification. Cys 306 is one of three cysteines that are unique to Bacillus glutamine synthetase. The result suggested that the cysteine has a role in the active site of the enzyme. We also report that the amino acid residue modified by FSBA was the lysyl residue at position 43.     

Catalytic cycle of the biosynthetic reaction catalyzed by adenylylated glutamine synthetase from Escherichia coli

The covalently attached AMP moiety of adenylylated glutamine synthetase from Escherichia coli has been replaced by its fluorescent analog, 2-aza-1,N6-etheno-AMP (aza-epsilon-AMP). The modified glutamine synthetase (aza-epsilon-GS) exhibits divalent cation requirement (Mn2+, rather than Mg2+), pH profile, Vmax, and Km similar to those of naturally adenylylated glutamine synthetase. Whereas naturally adenylylated glutamine synthetase exhibits only negligible fluorescence changes upon the binding of substrates, aza-epsilon-GS exhibits large fluorescence changes. The fluorescence changes have been used by means of a stopped flow technique to reveal the involvement of five fluorometrically distinct intermediates in the catalytic cycle for the biosynthesis of glutamine catalyzed by the adenylylated glutamine synthetase. The mechanism is very similar to that previously established for the unadenylylated enzyme, using intrinsic tryptophan fluorescence. Substrates bind via a rapid equilibrium random mechanism, but the reaction proceeds in a stepwise manner. The formation of an enzyme-bound intermediate (probably gamma-glutamyl phosphate + ADP) from ATP and L-glutamate is the rate-limiting step, with the subsequent reaction of the enzyme-bound intermediate occurring very rapidly. The success in elucidating this complex mechanism is due largely to the vastly different amplitudes of the fluorescence changes at the two excitation maxima (300 nm and 360 nm) of the aza-epsilon-AMP moiety which accompany the formation of the various intermediates.     

Escherichia coli gamma-glutamylcysteine synthetase. Two active site metal ions affect substrate and inhibitor binding.

Gamma-glutamylcysteine synthetase (gamma-GCS, glutamate-cysteine ligase), which catalyzes the first and rate-limiting step in glutathione biosynthesis, is present in many prokaryotes and in virtually all eukaryotes. Although all eukaryotic gamma-GCS isoforms examined to date are rapidly inhibited by buthionine sulfoximine (BSO), most reports indicate that bacterial gamma-GCS is resistant to BSO. We have confirmed the latter finding with Escherichia coli gamma-GCS under standard assay conditions, showing both decreased initial binding affinity for BSO and a reduced rate of BSO-mediated inactivation compared with mammalian isoforms. We also find that substitution of Mn2+ for Mg2+ in assay mixtures increases both the initial binding affinity of BSO and the rate at which BSO causes mechanism-based inactivation. Similarly, the specificity of E. coli gamma-GCS for its amino acid substrates is broadened in the presence of Mn2+, and the rate of reaction for some very poor substrates is improved. These results suggest that divalent metal ions have a role in amino acid binding to E. coli gamma-GCS. Electron paramagnetic resonance (EPR) studies carried out with Mn2+ show that E. coli gamma-GCS binds two divalent metal ions; Kd values for Mn2+ are 1.1 microm and 82 microm, respectively. Binding of l-glutamate or l-BSO to the two Mn2+/gamma-GCS species produces additional upfield and downfield X-band EPR hyperfine lines at 45 G intervals, a result indicating that the two Mn2+ are spin-coupled and thus apparently separated by 5 A or less in the active site. Additional EPR studies in which Cu2+ replaced Mg2+ or Mn2+ suggest that Cu2+ is bound by one N and three O ligands in the gamma-GCS active site. The results are discussed in the context of the catalytic mechanism of gamma-GCS and its relationship to the more fully characterized glutamine synthetase reaction.     

o-Phosphotyrosyl glutamine synthetase: modification of the nucleotide ligation site of adenylylated glutamine synthetase

The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes. The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase. The Km for this macromolecular substrate with the nuclease was 40 microM, at pH 8.9. The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased. The Km for the native adenylylated glutamine synthetase with the SVPDE was 36 microM, i.e., similar to that for the nuclease. When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme. Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme. The optimum pH, apparent Km for each of three substrates, glutamate, ATP, and NH3, and Vmax were in good agreement, as to either Mg2+- or Mn2+-dependent biosynthetic activity. From these results we can conclude that the regulation of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation.     

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+.     

Independent bindings of Mn2+ and Mg2+ to the active site of B. cereus glutamine synthetase

Glutamine synthetase purified from Bacillus cereus IFO 3131 was modified by iodoacetamide and the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). Only Mg2+-dependent activity was inactivated by iodoacetamide, whereas only Mn2+-dependent activity was inactivated by FSBA. When iodoacetamide-treated enzyme was reacted with FSBA, Mn2+-dependent activity was also inactivated. Mg2+ plus Mn2+-dependent activity was inactivated in any case. The results suggested that the binding sites of Mn2+ and Mg2+ are separate from each other in the active site of B. cereus glutamine synthetase and that bindings of Mg2+ and Mn2+ to each site are required for normal activity in vivo.