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 Förster 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 Förster-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.
Lin
W Y
WY
Department of Chemistry, Pennsylvania State University, University Park 16802.
Dombrosky
P
P
Atkins
W M
WM
Villafranca
J J
JJ
D001665:Binding Sites; D001974:Bromosuccinimide; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008163:Luminescent Measurements; D009154:Mutation; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry*; D014961:X-Ray Diffraction
D013725:Terbium; D014443:Tyrosine; D005974:Glutamate-Ammonia Ligase; D001974:Bromosuccinimide