Tryptophan-tRNA synthetase from beef pancreas. Characteristics of its intrinsic and extrinsic chromophores in relation to the conformation of the enzyme and its active site

Abstract

A simple and rapid method of obtaining, from beef pancreas, a homogeneous tryptophanyl‐tRNA synthetase without tryptophan, is described. Spectrophotometric and fluorimetric methods have provided new insights into the conformation of the enzyme in absence of substrate.By spectrophotometry, 26 tyrosine groups per mole of enzyme are titrated in the native protein. These chromophores present an abnormally high pK of 11.3 and also have a low accessibility to ethylene glycol and glycerol. In 8 M urea 29 tyrosines are titrated, compared to 36 residues found by amino acid analysis.By spectrofluorimetry, using a fluorescent probe of conformation, 1‐anilino‐8‐naphtalen sulfonate (ANS), we have demonstrated that there is one binding site for ANS per mole of enzyme. The association constant KA= 0.8 × 105 M–1 has been calculated for the complex ANS‐enzyme. The emission wavelength of the complex at 465 nm (instead of 515 nm for ANS in water) suggests a very hydrophobic environment for the bound ANS. The existence of energy transfer between intrinsic chromophores of the protein and ANS is demonstrated. The presence of tryptamine, a competitive inhibitor of the enzymatic reaction, quenches the fluorescence emission of the bound ANS.Although the emission fluorescence of the bound dye is affected by the presence of tryptamine, interpretation of the data demonstrates that the binding of ANS does not occur at the same site as tryptamine. This seems to imply that the catalytic site is not directly involved in the binding of ANS to tryptophanyl‐tRNA synthetase.