Molecular Dissection of a Critical Specificity Determinant within the Amino Acid Editing Domain of Leucyl-tRNA Synthetase

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• 作者：Richard S. Mursinna ; Keun Woo Lee ; James M. Briggs ; and Susan A. Martinis
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：January 13, 2004
• 年：2004
• 卷：43
• 期：1
• 页码：155 - 165
• 全文大小：595K
• 年卷期：v.43,no.1(January 13, 2004)
• ISSN：1520-4995

文摘

A highly conserved threonine residue marks the amino acid binding pocket within the editingactive site of leucyl-tRNA synthetases (LeuRSs). It is essential to substrate specificity for the Escherichiacoli enzyme in that it blocks the cognate leucine amino acid from binding in the hydrolytic editing activesite. We combined mutagenesis and computational approaches to elucidate the molecular role of the criticalside chain of this threonine residue. Removal of the terminal methyl group of the threonine side chain byreplacement with serine yielded a mutant LeuRS that hydrolyzes Leu-tRNA^Leu. Substitution of valine forthe conserved threonine conferred similar activities to the wild-type enzyme. However, an additionalsubstitution within the editing active site suggested synergistic interactions with the conserved threoninesite that significantly affected amino acid editing. On the basis of our combined biochemical andcomputational data, we propose that the threonine 252 side chain not only sterically hinders the cognatecharged leucine from binding for hydrolysis but also plays a critical role in maintaining an active sitegeometry that is required for the fidelity of LeuRS.