The C-terminal Extension of Yeast Seryl-tRNA Synthetase Affects Stability of the Enzyme and Its Substrate Affinity (original) (raw)
Related papers
Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 2000
The involvement of amino acids within the motif 2 loop of Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) in serine and ATP binding was demonstrated previously [B. Lenhard et al., J. Biol. Chem. 272 (1997) 1136–1141]. In our attempt to analyze the structural basis for the substrate specificity and to explore further the catalytic mechanism employed by S. cerevisiae SerRS, two new active site mutants, SerRS11 and SerRS12, were constructed. The catalytic effects of amino acid replacement at positions Lys287, Asp288 and Ala289 with purified wild-type and mutant seryl-tRNA synthetases were tested. The alteration of these semi-conserved amino acids interferes with tRNA-dependent optimization of serine recognition. Additionally, mutated enzymes SerRS11 (Lys287Thr, Asp288Tyr, Ala289Val) and SerRS12 (Lys287Arg) are less sensitive to inhibition by two competitive inhibitors: serine hydroxamate, an analogue of serine, and 5′-O-[N-(L-seryl)-sulfamoyl]adenosine, a stable analogue of aminoacyl adenylate, than the wild-type enzyme. SerRS mutants also display different activation kinetics for serine and serine hydroxamate, indicating that specificity toward the substrates is modulated by amino acid replacement in the motif 2 loop.
European journal of biochemistry / FEBS, 1976
By following the tryptophan fluorescence of yeast seryl-tRNA synthetase on addition of tRNA Ser it was observed that the number of binding sites for tRNA decreases from two to one with increasing temperature, ATP or KCl concentration. Concomitantly a considerable decrease of the apparent binding constant was observed. The variation in the number of binding sites is explained by the presence of at least one temperature and ionic strength sensitive binding site and one temperature and ionic strength independent binding site. Relaxation kinetic experiments revealed two binding processes: a fast one depending on tRNA concentration and ionic strength and a slow one, which appeared to be independent of tRNA concentration and ionic strength. Enzyme kinetic studies showed that the activity of seryl-tRNA synthetase strongly depends on the KCl concentration and exhibits a maximum at 0.2 M KCl. Based on the data from relaxation and enzyme kinetic experiments a model is suggested for the recogn...
Journal of Bacteriology, 1995
A mutant of Escherichia coli resistant to serine hydroxamate which has a large increase in K m for serine of seryl-tRNA synthetase is described. The mutant serS gene was cloned and sequenced and was found to contain a single-base-pair mutation, resulting in the substitution of the residue alanine 262 by valine in motif 2. The methyl side chain of alanine 262 is not exposed at the active site, and molecular modeling indicated that replacement of alanine 262 by valine does not significantly affect the configuration of amino acids at the active site. This finding suggests that the residue at this position may be involved in a conformational change (possibly induced by ATP binding) which is necessary for optimal binding of the cognate amino acid.
Molecular BioSystems, 2014
Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal's standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
Promoting the Formation of an Active Synthetase/tRNA Complex by a Nonspecific tRNA-binding Domain
Journal of Biological Chemistry, 2008
Previous studies showed that valyl-tRNA synthetase of Saccharomyces cerevisiae contains an N-terminal polypeptide extension of 97 residues, which is absent from its bacterial relatives, but is conserved in its mammalian homologues. We showed herein that this appended domain and its human counterpart are both nonspecific tRNA-binding domains (K d ϳ 0.5 M). Deletion of the appended domain from the yeast enzyme severely impaired its tRNA binding, aminoacylation, and complementation activities. This N-domain-deleted yeast valyl-tRNA synthetase mutant could be rescued by fusion of the equivalent domain from its human homologue. Moreover, fusion of the N-domain of the yeast enzyme or its human counterpart to Escherichia coli glutaminyl-tRNA synthetase enabled the otherwise "inactive" prokaryotic enzyme to function as a yeast enzyme in vivo. Different from the native yeast enzyme, which showed different affinities toward mixed tRNA populations, the fusion enzyme exhibited similar binding affinities for all yeast tRNAs. These results not only underscore the significance of nonspecific tRNA binding in aminoacylation, but also provide insights into the mechanism of the formation of aminoacyl-tRNAs. Aminoacyl-tRNA synthetases are a group of ancient enzymes, each of which catalyzes the attachment of a specific amino acid to its cognate tRNAs. Aminoacyl-tRNAs are then delivered by elongation factor-1 (EF-1) 3 to ribosomes for protein translation. In prokaryotes, there are typically 20 aminoacyl-tRNA synthetases, one for each amino acid (1-4). In eukaryotes, protein synthesis occurs not only in the cytoplasm, but also in organelles, such as mitochondria and chloroplasts (5). Thus, eukaryotes, such as yeast, commonly have two genes that encode distinct sets of proteins for each aminoacylation activity, one localized to the cytoplasm and the other to the mitochondria. Each set aminoacylates the isoaccepting tRNAs
Nucleic Acids Research, 1995
Escherichia coll seryl-tRNA synthetase (SerRS) is a homo-dlmeric class II aminoacyl-tRNA synthetase. Each subunlt Is composed of two distinct domains: the N-terminal domain Is a 60 A long, arm-like coiled coil structure built up of two antiparallel a-helices, whereas the C-termlnal domain, the catalytic core, is an a-p structure overlying a seven-stranded antiparallel p-sheet. Deletion of the arm-like domain (SerRS A35-97) does not affect the amino acid activation step of the reaction, but reduces aminoacylation activity by more than three orders of magnitude. In the present study, it was shown that the formation of heterodimers from two aminoacylation defective homodlmers, the N-terminal deletion and an active site mutant (SerRS E355Q), restored charging activity. The aminoacylation activity in a mixture containing the heterodimers was compared to that of solutions containing the same concentrations of homodlmer. The activity of the mixture was eight times higher than the activities of the homodlmer solutions, and reached 50% of the theoretical value that would be expected if 50% of the mixture was in the heterodlmer form and assuming that a heterodimer contains only one active site. These results are in full agreement with the structural analysis of E.coll SerRS complexed with Its cognate tRNA and provide functional evidence for the crossdimer binding of tRNA In solution.