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.     

Crystals of glutamine synthetase from Escherichia coli.

Further details are given of crystals of glutamine synthetase prepared from Escherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et al. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and packing considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetase preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are $\text{a = 140 ± 2}\text{A}\text{̊}$, and $\text{c = 148 ± 2}\text{A}\text{̊}$. The Laue symmetry group is $\text{3}\text{̄}$m P3₁.     

Conformation-specific monoclonal antibodies to glutamine synthetase in Escherichia coli

Glutamine synthetase from Escherichia coli is composed of 12 identical subunits and exists in various forms: unadenylylated, adenylylated, divalent cation bound (taut), and divalent cation free (relaxed). The relaxed dodecamer readily dissociates into monomers upon exposure to 1 M urea or pH 8.0. Glutamine synthetase can be inactivated irreversibly by oxidizing a particular histidine residue or by incubating with methionine sulfoximine and ATP. In order to establish hybridoma monoclones that produce antibodies capable of differentiating between different conformers of glutamine synthetase, homogeneous antibodies produced from 7 clones (10-76-1, 48-76-1, 68-2-1, 57-142-2, 72-104-1, 68-3-2, 57-8-1) were characterized for their binding specificity and effects on glutamine synthetase activity. Two antibodies (10-76-1, 48-76-1) bind only to the monomeric form, two antibodies (57-142-2, 68-3-2) bind only to the dodecameric forms (taut or relaxed) and the three others (68-2-1, 72-104-1, 57-8-1) bind to both forms. At a low antibody concentration, 68-3-2 binds preferentially to taut glutamine synthetase over oxidized glutamine synthetase. None of the 7 antibodies differentiates between unadenylylated and adenylylated form. Nevertheless, the gamma-glutamyltransferase activities of the resulting antibody-glutamine synthetase complexes were influenced variably depending upon the state of adenylylation and the divalent cation.     

Thermodynamics of active-site ligand binding to Escherichia coli glutamine synthetase

Active-site ligand interactions with dodecameric glutamine synthetase from Escherichia coli have been studied by calorimetry and fluorometry using the nonhydrolyzable ATP analogue 5'-adenylyl imidodiphosphate (AMP-PNP), L-glutamate, L-Met-(S)-sulfoximine, and the transition-state analogue L-Met-(S)-sulfoximine phosphate. Measurements were made with the unadenylylated enzyme at pH 7.1 in the presence of 100 mM KCl and 1.0 mM MnCl2, under which conditions the two catalytically essential metal ion sites per subunit are occupied and the stoichiometry of active-site ligand binding is equal to 1.0 equiv/subunit. Thermodynamic linkage functions indicate that there is strong synergism between the binding of AMP-PNP and L-Met-(S)-sulfoximine (delta delta G' = -6.4 kJ/mol). In contrast, there is a small antagonistic effect between the binding of AMP-PNP and L-glutamate (delta delta G' = +1.4 kJ/mol). Proton effects were negligible (less than or equal to 0.2 equiv of H+ release or uptake/mol) for the different binding reactions. The binding of AMP-PNP (or ATP) to the enzyme is entropically controlled at 303 K with delta H = +5.4 kJ/mol and delta S = +150 J/(K.mol). At 303 K, the binding of L-glutamate (delta H = -22.2 kJ/mol) or L-Met-(S)-sulfoximine [delta H = -45.6 kJ/mol with delta Cp approximately equal to -670 +/- 420 J/(K.mol)] to the AMP-PNP.Mn.enzyme complex is enthalpically controlled with opposing delta S values of -29 or -46 J/(K.mol), respectively. The overall enthalpy change is negative and the overall entropy change is positive for the simultaneous binding of AMP-PNP and L-glutamate or of AMP-PNP and L-Met-(S)-sulfoximine to the enzyme. For the binding of the transition-state analogue L-Met-(S)-sulfoximine phosphate (which inactivates the enzyme by blocking active sites), both enthalpic and entropic contributions also are favorable at 303 K [delta G' approximately equal to -109 and delta H = -54.8 kJ/mol of subunit and delta S approximately equal to +180 J/(K.mol)].     

Structure of the N-terminal domain of Escherichia coli glutamine synthetase adenylyltransferase

We report the crystal structure of the N-terminal domain of Escherichia coli adenylyltransferase that catalyzes the reversible nucleotidylation of glutamine synthetase (GS), a key enzyme in nitrogen assimilation. This domain (AT-N440) catalyzes the deadenylylation and subsequent activation of GS. The structure has been divided into three subdomains, two of which bear some similarity to kanamycin nucleotidyltransferase (KNT). However, the orientation of the two domains in AT-N440 differs from that in KNT. The active site of AT-N440 has been identified on the basis of structural comparisons with KNT, DNA polymerase beta, and polyadenylate polymerase. AT-N440 has a cluster of metal binding residues that are conserved in polbeta-like nucleotidyl transferases. The location of residues conserved in all ATase sequences was found to cluster around the active site. Many of these residues are very likely to play a role in catalysis, substrate binding, or effector binding.     

Inhibition of Escherichia coli glutamine synthetase by alpha- and gamma-substituted phosphinothricins

We have investigated the inhibition of Escherichia coli glutamine synthetase (GS) with alpha- and gamma-substituted analogues of phosphinothricin [L-2-amino-4-(hydroxymethylphosphinyl)butanoic acid (PPT)], a naturally occurring inhibitor of GS. These compounds display inhibition of bacterial GS that is competitive vs L-glutamate, with Ki values in the low micromolar range. At concentrations greater than Ki the phosphinothricins caused time-dependent loss of enzyme activity, while dilution after enzyme inactivation resulted in recovery of enzyme activity. ATP was required for inactivation; the nonhydrolyzable ATP analogue AMP-PCP failed to support inhibition of GS by the phosphinothricins. The binding of these inhibitors to the enzyme was also characterized by measurement of changes in protein fluorescence, which provided similar inactivation rate constants k1 and k2 for the entire series of compounds. Rate constants koff for recovery were also determined by fluorescence measurement and were comparable for both PPT and the gamma-hydroxylated analogue GHPPT and significantly greater for the alpha- and gamma-alkyl-substituted compounds. Electron paramagnetic resonance spectra provided information on the interaction of the phosphinothricins with the manganese form of the enzyme in the absence of ATP, and significant binding was observed for PPT and GHPPT. 31P NMR experiments confirmed that enzyme inactivation is accompanied by hydrolysis of ATP, although phosphorylated phosphinothricins could not be detected in solution. The kinetic behavior of these compounds is consistent with a mechanism involving inhibitor phosphorylation, followed by release from the active site and simultaneous hydrolysis to form Pi and free inhibitor.     

Subunit interaction in unadenylylated glutamine synthetase from Escherichia coli. Evidence from methionine sulfoximine inhibition studies

Although glutamine synthetase from Escherichia coli is composed of 12 identical subunits, there is no evidence that homologous subunit interactions occur in fully unadenylylated or fully adenylylated enzyme. Meister and co-workers (Manning, J. M., Moore, S., Rowe, W. B., and Meister, A. (1969) Biochemistry 8, 2681-2685) have shown that L-methionine-S-sulfoximine, one of the four diastereomers of methionine sulfoximine, preferentially inhibits glutamine synthetase irreversibly in the presence of ATP, due to the formation of tightly bound products, ADP, and methionine sulfoximine phosphate. Using highly purified unadenylylated glutamine synthetase and the two resolved diastereomers of L-methionine-S,R-sulfoximine, we have studied both the kinetics of glutamine synthetase inactivation in the presence of excess methionine sulfoximine and ATP, and the binding of methionine sulfoximine to the enzyme. The results reveal that (a) the apparent first order rate constant of irreversible inactivation by the S isomer decreases progressively from the expected first order rate, indicating that an inactivated subunit retards the reactivity of its neighboring subunits toward methionine sulfoximine and ATP; (b) the R isomer does not inactivate glutamine synthetase irreversibly in the presence of ATP; however, the R isomer is capable of protecting the enzyme temporarily from the irreversible inhibition by the S isomer; and (c) the binding of the S isomer monitored by changes in protein fluorescence exhibits an apparent negative cooperative binding isotherm, whereas the R isomer yields an apparent positive cooperative pattern.     

Protease So from Escherichia coli preferentially degrades oxidatively damaged glutamine synthetase

After oxidative damage (e.g. induced with iron, ascorbate, and oxygen), the inactivated glutamine synthetase is selectively hydrolyzed in extracts of Escherichia coli. We therefore tested if glutamine synthetase treated with this system is hydrolyzed preferentially by any of the known E. coli proteases. Protease So, a cytoplasmic serine protease, was found to degrade the oxidized form of glutamine synthetase to acid-soluble peptides 5-10 times faster than the native glutamine synthetase. Degradation of the oxidized glutamine synthetase was inhibited by EDTA and stimulated 5-10-fold by Mg2+, Ca2+, or Mn2+, even though casein hydrolysis by protease So is not affected by divalent cations. Apparently, these cations affect the conformation of this substrate, making it more susceptible to proteolytic attack. Protease Re, another cytoplasmic protease, also degrades preferentially the oxidized form of glutamine synthetase and seems to correspond to the glutamine synthetase-degrading activity recently described by Roseman and Levine [1987) J. Biol. Chem. 262, 2101-2110). However, it is much less active in this reaction than protease So. No other soluble E. coli protease, including Do, Ci, Mi, Fa, Pi, or the ATP-dependent proteases Ti and La (the lon product), appears to degrade this oxidized protein. These results suggest that protease So participates in the hydrolysis of oxidatively damaged proteins and that E. coli has multiple systems for degrading different types of aberrant proteins.