Strictly Conserved Lysine of Prolyl-tRNA Synthetase Editing Domain Facilitates Binding and Positioning of Misacylated tRNAPro

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• 作者：Thomas G. Bartholow ; Brianne L. Sanford ; Bach Cao ; Heidi L. Schmit ; James M. Johnson ; Jet Meitzner ; Sudeep Bhattacharyya ; Karin Musier-Forsyth ; Sanchita Hati
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：February 18, 2014
• 年：2014
• 卷：53
• 期：6
• 页码：1059-1068
• 全文大小：476K
• ISSN：1520-4995

文摘

To ensure high fidelity in translation, many aminoacyl-tRNA synthetases, enzymes responsible for attaching specific amino acids to cognate tRNAs, require proof-reading mechanisms. Most bacterial prolyl-tRNA synthetases (ProRSs) misactivate alanine and employ a post-transfer editing mechanism to hydrolyze Ala-tRNA^Pro. This reaction occurs in a second catalytic site (INS) that is distinct from the synthetic active site. The 2鈥?OH of misacylated tRNA^Pro and several conserved residues in the Escherichia coli ProRS INS domain are directly involved in Ala-tRNA^Pro deacylation. Although mutation of the strictly conserved lysine 279 (K279) results in nearly complete loss of post-transfer editing activity, this residue does not directly participate in Ala-tRNA^Pro hydrolysis. We hypothesized that the role of K279 is to bind the phosphate backbone of the acceptor stem of misacylated tRNA^Pro and position it in the editing active site. To test this hypothesis, we carried out pK_a, charge neutralization, and free-energy of binding calculations. Site-directed mutagenesis and kinetic studies were performed to verify the computational results. The calculations revealed a considerably higher pK_a of K279 compared to an isolated lysine and showed that the protonated state of K279 is stabilized by the neighboring acidic residue. However, substitution of this acidic residue with a positively charged residue leads to a significant increase in Ala-tRNA^Pro hydrolysis, suggesting that enhancement in positive charge density in the vicinity of K279 favors tRNA binding. A charge-swapping experiment and free energy of binding calculations support the conclusion that the positive charge at position 279 is absolutely necessary for tRNA binding in the editing active site.