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

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′-OH of misacylated tRNA^Pro and several conserved residues in the <i>Escherichia coli</i> 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 p<i>K</i>_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 p<i>K</i>_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

Investigation of intrinsic dynamics of enzymes involved in metabolic pathways using coarse-grained normal mode analysis

<p>Intrinsic dynamics of proteins are known to play important roles in their function. In particular, collective dynamics of a protein, which are defined by the protein’s overall architecture, are important in promoting the active site conformation that favors substrate binding and effective catalysis. The primary sequence of a protein, which determines its three-dimensional structure, encodes unique dynamics. The intrinsic dynamics of a protein actually link protein structure to its function. In the present study, coarse-grained normal mode analysis was performed to examine the intrinsic dynamic patterns of 24 different enzymes involved in primary metabolic pathways. We observed that each metabolic enzyme exhibits unique patterns of motions, which are conserved across multiple species and functionally relevant. Dynamic cross-correlation matrices (DCCMs) are visibly identical for a given enzyme family but significantly different from DCCMs of other protein families, reinforcing that proteins with similar function exhibit a similar pattern of motions. The present work also reasserted that correct identification of unknown proteins is possible based on their intrinsic mobility patterns.</p