Role of the Tign Sequence in Escherichia-Coli Tryptophanyl-Transfer-Rna Synthetase
by Chan, K. W.; Koeppe, R. E.
Tryptophanyl-tRNA synthetase in E. coli does not have the HIGH sequence that is normally characteristic of class I aminoacyl-tRNA synthetases (EC 22.214.171.124), but instead contains a TIGN sequence at residues 17-20, which has been suggested to be equivalent to the HIGH sequence (Jones, M.D. et al. (1986) Biochemistry 25, 1887-1891). We have overexpressed E. coli Trp-tRNA synthetase and have used site-directed mutagenesis to mutate Thr-17 in the TIGN sequence to alanine. The mutant enzyme has the same K-m values as the wild-type for tryptophan or tRNA(Trp), and a slightly increased K-m for ATP, from 0.37 to 0.64 mM. On the other hand, the k(cat) for either the first step or the overall reaction is decreased by a factor of 30. In comparing the Thr-17 and Ala-17 enzymes, the Delta Delta G for the conversion of substrate to transition state is + 9.6 kJ/mol (2.3 kcal/mol). Thr-17 is therefore important in binding the substrate in the transition state, thus supporting the suggestion that TIGN may fulfill the role of a HIGH sequence.
- Biochimica et Biophysica Acta-Protein Structure and Molecular Enzymology
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