Defining the active site of yeast seryl-tRNA synthetase. Mutations in motif 2 loop residues affect tRNA-dependent amino acid recognition.

The Journal of Biological Chemistry
B LenhardIvana Weygand-Durasevic

Abstract

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 Km values for serine and ATP, accompanied by decreases in kcat (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 allo...Continue Reading

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Citations

Dec 19, 2001·Archives of Biochemistry and Biophysics·Jasmina Rokov-PlavecIvana Weygand-Durasevic
Apr 29, 2006·Journal of Computer-aided Molecular Design·Sai Chetan K SukuruLeslie A Kuhn
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Oct 24, 2002·European Journal of Biochemistry·Ita Gruic-SovuljIvana Weygand-Durasevic
Jan 16, 1999·Nucleic Acids Research·B LenhardI Weygand-Durasević
Feb 7, 2004·European Journal of Biochemistry·Silvija BilokapicIvana Weygand-Durasevic
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Mar 21, 2017·Journal of Biomolecular Structure & Dynamics·Saheb DuttaNilashis Nandi
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