Ancient adaptation of the active site of tryptophanyl-tRNA synthetase for tryptophan binding.
The amino acid binding domains of the tryptophanyl (TrpRS)- and tyrosyl-tRNA synthetases (TyrRS) of Bacillus stearothermophilus are highly homologous. These similarities suggest that conserved residues in TrpRS may be responsible for both determining tryptophan recognition and discrimination against tyrosine. This was investigated by the systematic mutation of TrpRS residues based upon the identity of homologous positions in TyrRS. Of the four residues which interact directly with the aromatic side chain of tryptophan (Phe5, Met129, Asp132, and Val141) replacements of Asp132 led to significant changes in the catalytic efficiency of Trp aminoacylation (200-1250-fold reduction in k(cat)/K(M)) and substitution of Val141 by the larger Glu side chain reduced k(cat)/K(M) by 300-fold. Mutation of Pro127, which determines the position of active-site residues, did not significantly effect Trp binding. Of the mutants tested, D132N TrpRS also showed a significant reduction in discrimination against Tyr, with Tyr acting as a competitive inhibitor but not a substrate. The analogous residue in B. stearothermophilusTyrRS (Asp176) has also been implicated as a determinant of amino acid specificity in earlier studies [de Prat Gay, G., Duckworth, H. W., and Fersht, A. R. (1993) FEBS Lett. 318, 167-171]. This striking similarity in the function of a highly conserved residue found in both TrpRS and TyrRS provides mechanistic support for a common origin of the two enzymes.
Praetorius-Ibba
M
M
Department of Molecular Biophysics and Biochemistry, and Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8114, USA.
Stange-Thomann
N
N
Kitabatake
M
M
Ali
K
K
Söll
I
I
Carter
C W
CW
Ibba
M
M
Söll
D
D
D000215:Acylation; D000818:Animals; D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism; D019143:Evolution, Molecular; D001411:Geobacillus stearothermophilus / Q000235:genetics; D006801:Humans; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D012364:RNA, Transfer, Trp / Q000235:genetics; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*; D014369:Tryptophan-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011756:Diphosphates; D012364:RNA, Transfer, Trp; D014443:Tyrosine; D014364:Tryptophan; D014369:Tryptophan-tRNA Ligase