EPR investigation of the Mn(II) binding sites in glutamine synthetase (Escherichia coli W). II. Intermediate-affinity binding sites.

The nature of the intermediate-affinity (n2) Mn(II) binding sites in glutamine synthetase [EC 6.3.1.2] has been studied as a function of adenylylation in a variety of enzyme-metal complexes by EPR. In the absence of nucleotide the n2 Mn(II) environment is nearly isotropic, the Mn(II) bonds are highly ionic, and the interaction distance R greater than or equal to 12-14 A. Nucleotide binding at the n2 Mn(II) site renders the n2 Mn(II) signal unobservable and causes a reduction in signal amplitude (approximately 30%) and line broadening (approximately 6 G) at the high-affinity (n1) Mn(II) site. This behavior indicates that nucleotide binding induces a conformational change in the enzyme which brings the previously distant n1 and n2 sites into closer proximity (R less than or equal to 8-11 A), possibly for the purpose of activating the nucleotide for direct phosphoryl transfer to L-glutamate. In line with this suggestion, the broad, unresolved resonances in complexes containing both L-methionine SR-sulfoximine (MSOX) and nucleotide may result from the phosphorylation of MSOX. The n2 Mn(II) site is not affected by adenylylation in all the enzyme-metal complexes studied, which suggests that the regulatory effects of adenylylation may only act at the n1 Mn(II) sites.     

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.     

Terbium(III) luminescence study of the spatial relationship of tryptophan residues to the two metal ion binding sites of Escherichia coli glutamine synthetase.

The luminescence of Tb(III) was used to explore the topography of the metal ion sites of Escherichia coli glutamine synthetase and the relationship between these sites and tryptophan residues of the enzyme. By irradiation of tryptophan residues at 295 nm and measurement of the resulting Tb(III) luminescence at 544 nm, a biphasic curve was obtained upon titrating apoenzyme with Tb(III) indicating sequential binding of Tb(III) ions to the two binding sites of glutamine synthetase. The luminescence intensity was greater in the second region of the titration curve which is mostly due to energy transfer from Trp-158 to the second Tb(III) binding site of the enzyme. By use of the F?rster equation for energy transfer from donor Trp to acceptor Tb(III), distances from Trp-57 to Tb(III) at the n1 and n2 sites were calculated, by using a mutant enzyme in which Trp-158 was replaced by Ser, to be 16.4 and 15.7 A, respectively; distances from Trp-158 to Tb(III) at the n1 and n2 sites were calculated, by using a mutant enzyme in which Trp-57 was replaced by Leu, to be 16.8 and 9.5 A, respectively. All the distances are in reasonably good agreement with the crystal structure distances from Salmonella typhimurium glutamine synthetase except the distance from Trp-158 to the second Tb(III) binding site. The discrepancies may result from a slightly different conformation of glutamine synthetase in solution and in the crystal and/or a slightly different conformation for trivalent Ln(III) binding compared to divalent Mn(II) binding.     

Fluorescent probes for measuring the binding constants and distances between the metal ions bound to Escherichia coli glutamine synthetase.

TNS, 2-p-toluidinylnaphthalene-6-sulfonate, has been used as a fluorescent probe to determine the binding constants of metal ions to the two binding sites of Escherichia coli glutamine synthetase (GS). TNS fluorescence is enhanced dramatically when bound to proteins due to its high quantum yield resulting from its interactions with hydrophobic regions in proteins. The fluorescence energy transfer from a hydrophobic tryptophan residue of GS to TNS has been detected as an excitation band centered at 280 nm. Therefore, TNS is believed to be bound to a hydrophobic site on the GS surface other than the active site and is located near a hydrophobic Trp residue of GS. GS binds lanthanide ions [Ln(III)] more tightly than either Mn(II) or Mg(II), and the binding constants of several lanthanide ions were determined to be in the range (2.1-4.6) x 10(10) and (1.4-3.0) x 10(8) M-1 to the two metal binding sites of GS, respectively. The intermetal distances between the two metal binding sites of GS were also determined by measuring the efficiencies of energy transfer from Tb(III) to other Ln(III) ions. The intermetal distances of Tb(III)-Ho(III) and Tb(III)-Nd(III) were 7.9 and 6.8 A, respectively.     

Interacting binding sites of isoleucyl-tRNA synthetase from Escherichia coli studied by equilibrium partition.

The binding of tRNAIIe to isoleucyl-tRNA synthetase in the presence of isoleucine or ATP was investigated using the equilibrium partition method. Isoleucine decreased the affinity of tRNAIIe for the enzyme by a factor of about 5. For the free standard energy of interaction a value of about 1 kcal/mol (4.2 kJ/mol) was calculated. ATP exhibits qualitatively the same effect as isoleucine. A binding of two molecules isoleucine per molecule of enzyme could not be demonstrated even in the presence of ATP and pyrophosphatase.     

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.     

The sites of cleavage on Escherichia coli glutamine synthetase by dithiothreitol, Fe(III) and O2.

Native conformation of an enzyme molecule is required for the specific non-enzymatic cleavage of Escherichia coli glutamine synthetase by a metal-catalyzing oxidation system comprised of dithiothreitol, Fe(III) and O2. The cleavage reaction is greatly inhibited by the addition of Mg(II). Two major cleavage sites are identified between amino acid residues 264 and 268, and roughly between amino acid residues 31 and 34, which are located on the protein segments forming the active site of the enzyme. These results suggest that the cleavage reaction is a largely site-specific process involving active oxygen species generated at the divalent cation binding sites on glutamine synthetase.     

Terbium(III) luminescence study of tyrosine emission from Escherichia coli glutamine synthetase.

Radiationless energy transfer from tyrosine to Tb(III) in Escherichia coli glutamine synthetase and its two mutants (W57L and W158S) has been utilized to assess the tyrosine residue(s) responsible for the observed tyrosine emission and to investigate its spatial relationships to the two metal binding sites of GS. The interference from tryptophan fluorescence was removed by chemical modification of the tryptophan residues by N-bromosuccinimide (NBS). The Tyr-Tb(III) distances measured by using F?rster energy-transfer theory were in good agreement among the three enzymes with average distances of 10.7 and 11.2 A from Tyr to the two metal binding sites. The pKa value for the ionization of tyrosine was determined from fluorescence titration experiments to be approximately 10 for both mutant enzymes. The similarities in pKa values and Tyr-Tb(III) distances observed for all three enzymes lead to the conclusion that the same tyrosine residue(s), is (are) most likely responsible for the Tyr emission. According to the crystal structure distances from tyrosine residues to the two metal binding sites of GS, it is believed that Tyr-179 is the main contributor to the observed Tyr emission. The fact that an intense Tyr emission was observed for W57L GS but not for W158S GS indicates that Trp-57 is much more effective than Trp-158 in quenching the Tyr-179 emission probably through a F?rster-type energy transfer. Furthermore, modification of Trp-57 by NBS causes no significant increase in Tyr-179 emission while replacement of Trp-57 by leucine does. This may indicate that oxidized Trp-57 is also an effective quencher for Tyr-179 emission.