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 Förster 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.
McNemar
L S
LS
Department of Chemistry, Pennsylvania State University, University Park 16802.
Lin
W Y
WY
Eads
C D
CD
Atkins
W M
WM
Dombrosky
P
P
Villafranca
J J
JJ
D001665:Binding Sites; D003001:Cloning, Molecular; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry* / Q000235:genetics; D008163:Luminescent Measurements; D008670:Metals / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D012680:Sensitivity and Specificity; D012694:Serine / Q000235:genetics; D013725:Terbium / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics; D014961:X-Ray Diffraction
D008670:Metals; D013725:Terbium; D012694:Serine; D014364:Tryptophan; D005974:Glutamate-Ammonia Ligase