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 posttransla tional adenylylation on the metal binding sites of Escherichia coli gl utamine synthetase (GS). These studies revealed the presence of two la nthanide ion binding sites of GS of either adenylylation extrema. Indi vidual emission decay lifetimes were obtained in both H2O and D2O solv ent systems, allowing for the determination of the number of water mol ecules 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+ b ound to the nl site of unadenylylated enzyme, GS(0), and fully adenyly lated enzyme, GS(12), respectively, and that there are 2.6 +/- 0.5 wat er molecules coordinated to Eu3+ at site n2 for both GS(0) and GS(12). Energy transfer measurements between the lanthanide donor-acceptor pa ir Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 +/- 1.5 Angstrom. Distances between a Tb3+ ion at site n2 and tryptoph an residues were also performed with the use of single-tryptophan muta nt forms of E. coli GS. The dissociation constant for lanthanide ion b inding to site n1 was observed to decrease from K-d = 0.35 +/- 0.09 mu M for GS(0) to K-d = 0.06 +/- 0.02 mu M for GS(12). The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; K-d = 3.8 +/- 0.9 mu M and K-d = 2.6 +/- 0.7 mu M for GS(0) and GS(12), respectively. Competition experime nts indicate that Mn2+ affinity at site n1 decreases as a function of increasing adenylylation state, from K-d = 0.05 +/- 0.02 mu M for GS(0 ) to K-d = 0.35 +/- 0.09 mu M for GS(12). Mn2+ affinity at site n2 rem ains unchanged (K-d = 5.3 +/- 1.3 mu M for GS(0) and K-d = 4.0 +/- 1.0 mu M for GS(12)). The observed divalent metal ion affinities, which a re affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991. Biochemistry 3 0:1413-1418), supporting the hypothesis that adenylylation regulates G S by altering substrate and metal ion affinities.