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Volume 272, Number 2, Issue of January 10, 1997 pp. 1136-1141
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Defining the Active Site of Yeast Seryl-tRNA Synthetase
MUTATIONS IN MOTIF 2 LOOP RESIDUES AFFECT tRNA-DEPENDENT AMINO ACID RECOGNITION

(Received for publication, August 8, 1996, and in revised form, October 9, 1996)

Boris Lenhard ^§ , Sanda Filipi ^§ , Irena Landeka ^§ , Ivan krti ^§ , Dieter Söll ^¶ and Ivana Weygand-Duraevi ^§

From the ^¶ Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114,  Department of Chemistry, Faculty of Science, University of Zagreb, 10000 Zagreb, Croatia, and ^§ Rudjer Bokovi Institute, 10000 Zagreb, Croatia

The active site of class II aminoacyl-tRNA synthetases contains the motif 2 loop, which is involved in binding of ATP, amino acid, and the acceptor end of tRNA. In order to characterize the active site of Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS), we performed in vitro mutagenesis of the portion of the SES1 gene encoding the motif 2 loop. Substitutions of amino acids conserved in the motif 2 loop of seryl-tRNA synthetases from other sources led to loss of complementation of a yeast SES1 null allele strain by the mutant yeast SES1 genes. Steady-state kinetic analyses of the purified mutant SerRS proteins revealed elevated K_m values for serine and ATP, accompanied by decreases in k_cat (as expected for replacement of residues involved in aminoacyl-adenylate formation). The differences in the affinities for serine and ATP, in the absence and presence of tRNA are consistent with the proposed conformational changes induced by positioning the 3-end of tRNA into the active site, as observed recently in structural studies of Thermus thermophilus SerRS (Cusack, S., Yaremchuk, A., and Tukalo, M. (1996) EMBO J. 15, 2834-2842). The crystal structure of this moderately homologous prokaryotic counterpart of the yeast enzyme allowed us to produce a model of the yeast SerRS structure and to place the mutations in a structural context. In conjunction with structural data for T. thermophilus SerRS, the kinetic data presented here suggest that yeast seryl-tRNA synthetase displays tRNA-dependent amino acid recognition.

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