Defining the active site of yeast seryl-tRNA synthetase. Mutations in motif 2 loop residues affect tRNA-dependent amino acid recognition.
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 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.
Lenhard
B
B
Department of Chemistry, Faculty of Science, University of Zagreb, Croatia.
Filipić
S
S
Landeka
I
I
Skrtić
I
I
Söll
D
D
Weygand-Durasević
I
I
D000595:Amino Acid Sequence; D001665:Binding Sites; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011487:Protein Conformation; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D012714:Serine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus
D012714:Serine-tRNA Ligase