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.The formation of aminoacyl-tRNA, catalyzed by aminoacyltRNA synthetases, is a crucial step in maintaining the fidelity of protein biosynthesis. This family of enzymes can be partitioned into two classes of 10 enzymes each, based on conserved sequences (1) and structural motifs (2). All members of class I contain a common loop with the signature sequence KMSKS (3) and a region of homology with the HIGH peptide (4) as part of a Rossmann dinucleotide binding fold of parallel -sheets (5). Class II synthetases have a different topology of dinucleotide binding based on antiparallel -sheets (2, 6, 7). The three common signature motifs of class II synthetases are found in this domain. Motif 1 forms part of the conserved inter-subunit interface of homodimeric (6, 8) and heteromeric (9) synthetases. Motifs 2 and 3 contain many of the active-site residues important for ATP, amino acid, and tRNA acceptor stem recognition (10 -15). The elucidation of the crucial role of sequence motifs in substrate binding have resulted from the solution of several crystal structures of enzymes and enzyme-substrate complexes from both class I and class II (16) and numerous biochemical studies involving mutant synthetases (17-22).The evolution of tRNA recognition systems has recently gained much attention (23-27). The primary structure of several prokaryotic and eukaryotic seryl-tRNA synthetases (Ref.23; see also the legends to Fig. 1 and 3), including the enzyme that probably functions in yeast mitochondria (28), have been determi...