The enzymatic post-transfer editing mechanism of
e. coli threonyl-trna synthetase (thrrs): an md and qm/mm investigation
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Threonyl-tRNA synthetase (ThrRS) is a class II aminoacyl-tRNA synthetase (aaRS), which are a ubiquitous family of enzymes that have a vital role in protein biosynthesis. In particular, it catalyzes the activation and subsequent
aminoacylation of its corresponding tRNAThr. Because of the close structural and electronic similarity between its cognate substrate threonine and the noncognate serine, the catalytic aminoacylation site of ThrRS is not able to fully discriminate between them. In this study we have explored multiple possible post-transfer editing mechanisms for ThrRS from Escherichia coli.
The editing site is known to contain two conserved histidyls (His73 and His186) and a cysteinyl (Cys182), all of which could act as the required mechanistic base. We have performed detailed molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) studies in which the protonation states of each of these residues was varied.
Furthermore, using the various substrate-bound active site models obtained, we have examined previously proposed and alternative possible mechanisms for deaminoacylation of Ser-tRNAThr by ThrRS in which His73 or Cys182 acts as the base:
11 mechanisms in total. The present results suggest that the most feasible mechanism is obtained when both His73 and His186 are neutral, while the thiol of Cys182 is deprotonated and acts as a base. The resulting mechanism is found to occur in two steps. First, deprotonation of an active site water by the thiolate of Cys182 with its concomitant nucleophilic attack at the substrate’s Ccarbcenter occurs with a calculated free energy barrier of 9.9 kcal/mol. The subsequent, and overall rate-limiting step, is a water-meditated proton transfer from Lys156 onto the Ado763′-oxygen resulting in simultaneous cleavage of the Ado763′O–Ccarb bond with a free energy barrier of 20.8 kcal/mol.
I Hope It's help you....!!!!
_____________
_____________
Threonyl-tRNA synthetase (ThrRS) is a class II aminoacyl-tRNA synthetase (aaRS), which are a ubiquitous family of enzymes that have a vital role in protein biosynthesis. In particular, it catalyzes the activation and subsequent
aminoacylation of its corresponding tRNAThr. Because of the close structural and electronic similarity between its cognate substrate threonine and the noncognate serine, the catalytic aminoacylation site of ThrRS is not able to fully discriminate between them. In this study we have explored multiple possible post-transfer editing mechanisms for ThrRS from Escherichia coli.
The editing site is known to contain two conserved histidyls (His73 and His186) and a cysteinyl (Cys182), all of which could act as the required mechanistic base. We have performed detailed molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) studies in which the protonation states of each of these residues was varied.
Furthermore, using the various substrate-bound active site models obtained, we have examined previously proposed and alternative possible mechanisms for deaminoacylation of Ser-tRNAThr by ThrRS in which His73 or Cys182 acts as the base:
11 mechanisms in total. The present results suggest that the most feasible mechanism is obtained when both His73 and His186 are neutral, while the thiol of Cys182 is deprotonated and acts as a base. The resulting mechanism is found to occur in two steps. First, deprotonation of an active site water by the thiolate of Cys182 with its concomitant nucleophilic attack at the substrate’s Ccarbcenter occurs with a calculated free energy barrier of 9.9 kcal/mol. The subsequent, and overall rate-limiting step, is a water-meditated proton transfer from Lys156 onto the Ado763′-oxygen resulting in simultaneous cleavage of the Ado763′O–Ccarb bond with a free energy barrier of 20.8 kcal/mol.
I Hope It's help you....!!!!
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