UVM Theses and Dissertations
Format:
Print
Author:
Minajigi, Anand
Dept./Program:
Cell and Molecular Biology Program
Year:
2010
Degree:
Ph. D.
Abstract:
Aminoacyl-tRNA synthetases (ARSs) maintain the fidelity of protein synthesis by attaching a specific amino acid to its corresponding tRNA with the correct anticodon. Mistakes in aminoacylation can lead to molecular pathologies. The editing mechanisms exhibited by ARSs allow for correction of mistakes. In this dissertation, a mechanistic investigation of aminoacylation and editing by E. coli threonyl-tRNA synthetase (ThrRS) was carried out employing pre-steady state rapid kinetic techniques. Similar to other class II ARSs, amino acid activation in the presence of tRNA is the rate-limiting step of overall aminoacylation.
In contrast to class II histidyl-tRNA synthetase, the pro-Sp oxygen of adenylate is not the general base for the aminoacyl transfer in ThrRS. Instead, the 2'-OH of tRNA A76 and conserved His309 collaborate to activate the 3'-OH of tRNA A76 for aminoacyl-transfer. Based on this data, a proton-relay mechanism is proposed for aminoacyl-transfer reaction in ThrRS that is reminiscent of the NAD-dependent mechanisms of alcohol dehydrogenases, and the RNA-mediated catalysis of the ribosomal peptidyl transferase center. In the presence of the near-cognate amino acid serine, significant pre-transfer editing occurs via enzyme mediated adenylate hydrolysis. Asymmetric catalysis is present in activation of both threonine and serine. Amino acid specificity is not exhibited during the aminoacyl-transfer reaction.
An energy efficiency analysis of ThrRS clearly demonstrated that the rate of aminoacyl transfer dictates choice of editing pathway. Determination of inhibition profile of ThrRS by borrelidin and its analogues helped to identify a highly potent analogue with an ICso in the pM range. In summary, the pre-steady state investigation of E. coli ThrRS uncovered the similarities and differences in the kinetic mechanisms of ARSs at large, and provides a platform for analysis of anti-ARS drug action.
In contrast to class II histidyl-tRNA synthetase, the pro-Sp oxygen of adenylate is not the general base for the aminoacyl transfer in ThrRS. Instead, the 2'-OH of tRNA A76 and conserved His309 collaborate to activate the 3'-OH of tRNA A76 for aminoacyl-transfer. Based on this data, a proton-relay mechanism is proposed for aminoacyl-transfer reaction in ThrRS that is reminiscent of the NAD-dependent mechanisms of alcohol dehydrogenases, and the RNA-mediated catalysis of the ribosomal peptidyl transferase center. In the presence of the near-cognate amino acid serine, significant pre-transfer editing occurs via enzyme mediated adenylate hydrolysis. Asymmetric catalysis is present in activation of both threonine and serine. Amino acid specificity is not exhibited during the aminoacyl-transfer reaction.
An energy efficiency analysis of ThrRS clearly demonstrated that the rate of aminoacyl transfer dictates choice of editing pathway. Determination of inhibition profile of ThrRS by borrelidin and its analogues helped to identify a highly potent analogue with an ICso in the pM range. In summary, the pre-steady state investigation of E. coli ThrRS uncovered the similarities and differences in the kinetic mechanisms of ARSs at large, and provides a platform for analysis of anti-ARS drug action.