PMID:12974627
| Citation |
Zhang, CM, Perona, JJ and Hou, YM (2003) Amino acid discrimination by a highly differentiated metal center of an aminoacyl-tRNA synthetase. Biochemistry 42:10931-7 |
|---|---|
| Abstract |
Escherichia coli cysteinyl-tRNA synthetase (CysRS) achieves high amino acid specificity without the need for an editing reaction. Crystallographic and spectroscopic studies have previously demonstrated that a major determinant of the specificity is an active site zinc ion that recognizes the substrate cysteine through a strong zinc-thiolate interaction. The active site cleft of CysRS is composed of highly or strictly conserved amino acids, including four inner-sphere zinc ligands, five histidine imidazoles at the base of the cleft, and a tryptophan that flips down upon cysteine binding to complete formation of the binding pocket. Here we establish the significance of each of these major features of the active site cleft by mutational analysis. Substitutions generally lead to substantially deleterious effects on K(m) and k(cat) parameters with respect to each of the cysteine, ATP, and tRNA(Cys) substrates. These findings emphasize the importance of the highly differentiated nature of the active site and provide new insights into the origins of selectivity without editing. Most mutants are less attenuated in tRNA aminoacylation than in adenylate synthesis, suggesting that tRNA binding drives a conformational change to help assemble the active site. |
| Links |
PubMed Online version:10.1021/bi034812u |
| Keywords |
Adenosine Triphosphate/chemistry; Amino Acids/chemistry; Amino Acyl-tRNA Synthetases/chemistry; Binding Sites; Circular Dichroism; Crystallography, X-Ray; Cysteine/chemistry; Escherichia coli/enzymology; Escherichia coli/metabolism; Histidine/chemistry; Imidazoles/chemistry; Kinetics; Ligands; Models, Molecular; Mutation; Protein Binding; Protein Conformation; RNA, Transfer/chemistry; Zinc/chemistry |
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