Aminoacylation of tRNAs as critical step of protein biosynthesis.
Isoleucyl-tRNA synthetases isolated from commercial baker's yeast and E coli were investigated for their sequences of substrate additions and product releases. The results show that aminoacylation of tRNA is catalyzed by these enzymes in different pathways, eg isoleucyl-tRNA synthetase from yeast can act with four different catalytic cycles. Amino acid specificities are gained by a four-step recognition process consisting of two initial binding and two proofreading steps. Isoleucyl-tRNA synthetase from yeast rejects noncognate amino acids with discrimination factors of D = 300-38000, isoleucyl-tRNA synthetase from E coli with factors of D = 600-68000. Differences in Gibbs free energies of binding between cognate and noncognate amino acids are related to different hydrophobic interaction energies and assumed conformational changes of the enzyme. A simple hypothetical model of the isoleucine binding site is postulated. Comparison of gene sequences of isoleucyl-tRNA synthetase from yeast and E coli exhibits only 27% homology. Both genes show the 'HIGH'- and 'KMSKS'-regions assigned to binding of ATP and tRNA. Deletion of 250 carboxyterminal amino acids from the yeast enzyme results in a fragment which is still active in the pyrophosphate exchange reaction but does not catalyze the aminoacylation reaction. The enzyme is unable to catalyze the latter reaction if more than 10 carboxyterminal residues are deleted.
Cramer
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F
Max-Planck-Institut für experimentelle Medizin, Abteilung Chemie, Göttingen, Germany.
Englisch
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U
Freist
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W
Sternbach
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H
D004926:Escherichia coli / Q000378:metabolism; D005800:Genes, Fungal; D007533:Isoleucine-tRNA Ligase / Q000235:genetics / Q000378:metabolism; D008967:Molecular Biology; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000378:metabolism; D012331:RNA, Fungal / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D012329:RNA, Bacterial; D012331:RNA, Fungal; D012346:RNA, Transfer, Amino Acyl; D007533:Isoleucine-tRNA Ligase