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  • Kinetic partitioning between synthetic and editing pathways in class I aminoacyl-tRNA synthetases occurs at both pre-transfer and post-transfer hydrolytic steps.

Kinetic partitioning between synthetic and editing pathways in class I aminoacyl-tRNA synthetases occurs at both pre-transfer and post-transfer hydrolytic steps.

The Journal of biological chemistry (2012-06-01)
Nevena Cvetesic, John J Perona, Ita Gruic-Sovulj
ABSTRACT

Comprehensive steady-state and transient kinetic studies of the synthetic and editing activities of Escherichia coli leucyl-tRNA synthetase (LeuRS) demonstrate that the enzyme depends almost entirely on post-transfer editing to endow the cell with specificity against incorporation of norvaline into protein. Among the three class I tRNA synthetases possessing a dedicated post-transfer editing domain (connective peptide 1; CP1 domain), LeuRS resembles valyl-tRNA synthetase in its reliance on post-transfer editing, whereas isoleucyl-tRNA synthetase differs in retaining a distinct tRNA-dependent synthetic site pre-transfer editing activity to clear noncognate amino acids before misacylation. Further characterization of the post-transfer editing activity in LeuRS by single-turnover kinetics demonstrates that the rate-limiting step is dissociation of deacylated tRNA and/or amino acid product and highlights the critical role of a conserved aspartate residue in mediating the first-order hydrolytic steps on the enzyme. Parallel analyses of adenylate and aminoacyl-tRNA formation reactions by wild-type and mutant LeuRS demonstrate that the efficiency of post-transfer editing is controlled by kinetic partitioning between hydrolysis and dissociation of misacylated tRNA and shows that trans editing after rebinding is a competent kinetic pathway. Together with prior analyses of isoleucyl-tRNA synthetase and valyl-tRNA synthetase, these experiments provide the basis for a comprehensive model of editing by class I tRNA synthetases, in which kinetic partitioning plays an essential role at both pre-transfer and post-transfer steps.

MATERIALS
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Sigma-Aldrich
DL-Norvaline
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1 vial
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Sigma-Aldrich
L-Norvaline, arginase inhibitor
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1 vial
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$338.00
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