Amino acid-dependent transfer RNA affinity in a class I aminoacyl-tRNA synthetase.
Steady-state and transient kinetic analyses of glutaminyl-tRNA synthetase (GlnRS) reveal that the enzyme discriminates against noncognate glutamate at multiple steps during the overall aminoacylation reaction. A major portion of the selectivity arises in the amino acid activation portion of the reaction, whereas the discrimination in the overall two-step reaction arises from very weak binding of noncognate glutamate. Further transient kinetics experiments showed that tRNA(Gln) binds to GlnRS approximately 60-fold weaker when noncognate glutamate is present and that glutamate reduces the association rate of tRNA with the enzyme by 100-fold. These findings demonstrate that amino acid and tRNA binding are interdependent and reveal an important additional source of specificity in the aminoacylation reaction. Crystal structures of the GlnRS x tRNA complex bound to either amino acid have previously shown that glutamine and glutamate bind in distinct positions in the active site, providing a structural basis for the amino acid-dependent modulation of tRNA affinity. Together with other crystallographic data showing that ligand binding is essential to assembly of the GlnRS active site, these findings suggest a model for specificity generation in which required induced-fit rearrangements are significantly modulated by the identities of the bound substrates.
Uter
Nathan T
NT
Interdepartmental Program in Biomolecular Science and Engineering, University of California, Santa Barbara, California 93106-9510, USA.
Gruic-Sovulj
Ita
I
Perona
John J
JJ
D000596:Amino Acids / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism*
D000596:Amino Acids; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase