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 H₂0 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 Å. 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 nl was observed to decrease from K_d = 0.35 0.09 μM for GS₀ to K_d = 0.06 0.02 μM for GS₁₂. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; K_d = 3.8 0.9 μM and K_d = 2.6 0.7 μM for GS₀ and GS₁₂, respectively. Competition experiments indicate that Mn²⁺ affinity at site n1 decreases as a function of increasing adenylylation state, from K_d = 0.05 0.02 μM for GS₀ to K_d = 0.35 0.09 μM for GS₁₂. Mn²⁺ affinity at site n2 remains unchanged (K_d = 5.3 1.3 μM for GS₀ and K_d = 4.0 1.0 μM 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.