Kinetic partitioning between synthetic and editing pathways in class I aminoacyl-tRNA synthetases occurs at both pre-transfer and post-transfer hydrolytic steps.
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.
Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia.
Nevena
N
Cvetesic
John J
JJ
Perona
Ita
I
Gruic-Sovulj
D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D007935:Leucine-tRNA Ligase / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012356:RNA, Transfer, Leu / Q000378:metabolism*; D014633:Valine / Q000031:analogs & derivatives / Q000378:metabolism
D012346:RNA, Transfer, Amino Acyl; D012356:RNA, Transfer, Leu; C005313:norvaline; D007935:Leucine-tRNA Ligase; D014633:Valine