DEFINING THE ACTIVE-SITE OF YEAST SERYL-TRANSFER-RNA SYNTHETASE - MUTATIONS IN MOTIF-2 LOOP RESIDUES AFFECT TRANSFER-RNA-DEPENDENT AMINO-ACID RECOGNITION

The active site of class II aminoacyl-tRNA synthetases contains the mo tif 2 loop, which is involved in binding of ATP, amino acid, and the a cceptor end of tRNA. In order to characterize the active site of Sacch aromyces cerevisiae seryl-tRNA synthetase (SerRS), we performed in vit ro mutagenesis of the portion of the SES1 gene encoding the motif 2 lo op, Substitutions of amino acids conserved in the motif 2 loop of sery l-tRNA synthetases from other sources led to loss of complementation o f a yeast SES1 null allele strain by the mutant yeast SES1 genes. Stea dy-state kinetic analyses of the purified mutant SerRS proteins reveal ed 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 an d ATP, in the absence and presence of tRNA are consistent with the pro posed conformational changes induced by positioning the 3'-end of tRNA into the active site, as observed recently in structural studies of T hermus 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 mutation s 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.