Investigating Catalytically Important in Escherichia coli Prolyl-tRNA Synthetase Using SiteDirected Mutagenesis and Computational Studies
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This research project is a continuing effort focused on the molecular-level understanding of the relationship between structure, dynamics, and function in aminoacyl-tRNA synthetases (AARSs). Recent computational studies have identified four charged amino acid residues at the active site [three positively charged arginine (R) and one negatively charged glutamic acid (E)] that are directly involved in catalysis in Escherichia coli Prolyl-tRNA Synthetase (Ec ProRS). Our current objective is to experimentally confirm these computational findings by mutating these residues to a neutral amino acid, alanine, as well as two of the amino acids (E111 and R450) to aspartic acid, a shorter negatively charged amino acid. These mutations are also being run computationally in order to compare results and validate our computational model used. By studying the kinetics and catalytic rate of these mutations, we can determine the importance of these specific active site residues, indicated to be directly involved in the enzymatic ProRS reaction. This knowledge can potentially be used to develop selective drug against pathogenic AARSs. AARSs are potential drug targets as they play a key role in protein synthesis in all living organisms. Herein, we will present the preliminary result of our work.