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J. Biol. Chem., Vol. 280, Issue 25, 23966-23977, June 24, 2005

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Amino Acid-dependent Transfer RNA Affinity in a Class I Aminoacyl-tRNA Synthetase^*

Nathan T. Uter, Ita Gruic-Sovulj, and John J. Perona¶||

From the ^¶Department of Chemistry and Biochemistry, and Interdepartmental Program in Biomolecular Science and Engineering, University of California, Santa Barbara, California 93106-9510

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 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·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.

Received for publication, December 20, 2004 , and in revised form, April 18, 2005.

^* This work was supported by Grant GM63713 from the National Institutes of Health (to J. J. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Biochemistry Faculty of Science, University of Zagreb, Strossmayerov trg. 14, 10000 Zagreb, Croatia.

^|| To whom correspondence should be addressed: Dept. of Chemistry & Biochemistry, University of California, Santa Barbara, CA 93106-9510. Tel.: 805-893-7389; Fax: 805-893-4120; E-mail: perona{at}chem.ucsb.edu

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