Role of the TIGN sequence in E. coli tryptophanyl-tRNA synthetase.
Tryptophanyl-tRNA synthetase in E. coli does not have the HIGH sequence that is normally characteristic of class I aminoacyl-tRNA synthetases (EC 6.1.1.2), 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 Km values as the wild-type for tryptophan or tRNA(Trp), and a slightly increased Km for ATP, from 0.37 to 0.64 mM. On the other hand, the kcat 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.
Chan
K W
KW
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701.
Koeppe
R E
RE
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012364:RNA, Transfer, Trp / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013912:Threonine / Q000235:genetics; D014369:Tryptophan-tRNA Ligase / Q000235:genetics / Q000378:metabolism*
D012364:RNA, Transfer, Trp; D011994:Recombinant Proteins; D013912:Threonine; D014369:Tryptophan-tRNA Ligase