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Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy

1996; Wiley; Volume: 5; Issue: 12 Linguagem: Inglês

10.1002/pro.5560051216

1469-896X

Luis P. Reynaldo, Joseph J. Villafranca, William DeW. Horrocks,

Enzyme Structure and Function

Abstract 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 H 2 0 and D 2 O solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu 3+ . The results indicate that there are 4.3 0.5 and 4.6 0.5 water molecules coordinated to Eu 3+ bound to the n1 site of unadenylylated enzyme, GS 0 , and fully adenylylated enzyme, GS 12 , respectively, and that there are 2.6 0.5 water molecules coordinated to Eu 3+ at site n2 for both GS 0 and GS 12 . Energy transfer measurements between the lanthanide donor‐acceptor pair Eu 3+ and Nd 3+ , obtained an intermetal distance measurement of 12.1 1.5 Å. Distances between a Tb 3+ 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 GS 0 to K d = 0.06 0.02 μM 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 μM and K d = 2.6 0.7 μM for GS 0 and GS 12 , respectively. Competition experiments indicate that Mn 2+ affinity at site n1 decreases as a function of increasing adenylylation state, from K d = 0.05 0.02 μM for GS 0 to K d = 0.35 0.09 μM for GS 12 . Mn 2+ affinity at site n2 remains unchanged ( K d = 5.3 1.3 μM for GS 0 and K d = 4.0 1.0 μM for GS 12 ). 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.