PMID:14705941
| Citation |
Mursinna, RS, Lee, KW, Briggs, JM and Martinis, SA (2004) Molecular dissection of a critical specificity determinant within the amino acid editing domain of leucyl-tRNA synthetase. Biochemistry 43:155-65 |
|---|---|
| Abstract |
A highly conserved threonine residue marks the amino acid binding pocket within the editing active site of leucyl-tRNA synthetases (LeuRSs). It is essential to substrate specificity for the Escherichia coli enzyme in that it blocks the cognate leucine amino acid from binding in the hydrolytic editing active site. We combined mutagenesis and computational approaches to elucidate the molecular role of the critical side chain of this threonine residue. Removal of the terminal methyl group of the threonine side chain by replacement with serine yielded a mutant LeuRS that hydrolyzes Leu-tRNA(Leu). Substitution of valine for the conserved threonine conferred similar activities to the wild-type enzyme. However, an additional substitution within the editing active site suggested synergistic interactions with the conserved threonine site that significantly affected amino acid editing. On the basis of our combined biochemical and computational data, we propose that the threonine 252 side chain not only sterically hinders the cognate charged leucine from binding for hydrolysis but also plays a critical role in maintaining an active site geometry that is required for the fidelity of LeuRS. |
| Links |
PubMed Online version:10.1021/bi034919h |
| Keywords |
Amino Acid Substitution/genetics; Binding Sites/genetics; Computational Biology/methods; DNA Mutational Analysis; Enzyme Stability/genetics; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Hydrolysis; Leucine-tRNA Ligase/chemistry; Leucine-tRNA Ligase/genetics; Leucine-tRNA Ligase/metabolism; Mutagenesis, Site-Directed; Protein Binding/genetics; Protein Structure, Tertiary/genetics; RNA Editing/genetics; Serine/genetics; Substrate Specificity/genetics; Surface Properties; Threonine/genetics; Valine/genetics |
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