Structure-activity relationships in engineered proteins: characterization of disruptive deletions in the alpha-ammonium group binding site of tyrosyl-tRNA synthetase.
Residues Asp-78 and Gln-173 of the tyrosyl-tRNA synthetase of Bacillus stearothermophilus form part of the binding site for tyrosine by making hydrogen bonds with the alpha-ammonium group. Asp-38 is close enough to the group to make an important electrostatic contribution. Unlike other residues in the active site that have been studied by site-directed mutagenesis, Asp-38, Asp-78, and Gln-173 are part of hydrogen-bonded networks. Each of these residues has been mutated to an alanine, and the resultant mutants have been studied by kinetics to construct the difference energy diagrams for the formation of tyrosyl adenylate. In each example, the binding of tyrosine is weakened by about 2.5 kcal mol-1. But, unlike previous mutants, the dissociation of the second substrate, in this case ATP, is also seriously affected, being weakened by some 2 kcal mol-1 for TyrTS(Ala-78) and TyrTS(Ala-173). The energy of the transition state for the formation of tyrosyl adenylate is raised by 7.8 kcal mol-1 for the former and 4.5 kcal mol-1 for the latter mutant. Addition of these mutants to linear free energy plots constructed for the nondisruptive mutants in the accompanying study [Fersht, A. R., Leatherbarrow, R. J., & Wells, T. N. C. (1987) Biochemistry (preceding paper in this issue)] reveals large deviations of the data for TyrTS(Ala-38) and TyrTS(Ala-78) from the regression line. These thus belong to a different class of mutations from previous nondisruptive examples. This observation combined with the structural evidence and difference energy diagrams strongly suggests that the mutations Asp----Ala-38 and Asp----Ala-78 are disruptive in nature.
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D001483:Base Sequence; D002872:Chromosome Deletion; D005818:Genetic Engineering; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014448:Tyrosine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism
D011506:Proteins; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase