Anticooperative binding of L-tryptophan to tryptophanyl-tRNA synthetase from beef pancreas. Study at equilibrium by dialysis and changes in spectroscopic properties.

Equilibrium dialysis and gel filtration studies show that tryptophanyl-tRNA synthetase from beef pancreas binds two molecules of L-tryptophan per dimer in an anticooperative way. The binding of tryptophan ellicits a series of spectroscopic changes in the protein as seen by absorbance, fluorescence and circular dichroism. The molar absorption change of the protein-tryptophan system upon formation of the complex is delta epsilon292 = 10 400 +/- 1000 M(-1) cm(-1) per dimer. Taking an initial symmetrical dimeric protein the two dissociation constants for tryptophan at pH 8, 25 degrees C are respectively K1 = 2.0 +/- 0.5 muM and K2 = 10 +/- 4 muM. They are respectively K1 = 1 +/- 0.25 muM and K2 = 20 +/- 8 muM if one considers a sequenced binding of the two tryptophan molecules. The dichroic band at 290 nm of the free protein disappears when tryptophan is bound. All observed changes are characteristic of tryptophan perturbation and none of tyrosine perturbation. They all exceed the effect that can be expected from the change in environment of the bound tryptophan molecules and modifications of the tertiary structure of the protein have to be taken into account to explain the observed spectroscopic data.