Fluorescence Studies on Substrate Binding to Seryl‐tRNA Synthetase from Yeast

The interactions of seryl‐tRNA synthetase with its ligands have been investigated by fluorescence measurements. Using a microscale fluorescence technique and an excitation wavelength of 303 nm the inner filter effect was suppressed. The fluorescence intensity of the seryl‐tRNA synthetase at 342 nm (maximum emission) was quenched by 33%, by adding tRNASer, whereas no effect was produced by the tRNAs which are specific for phenylalanine and tyrosine. For tRNASer an association constant of 2.1 × 106 M−1 per enzyme subunit was calculated from the fluorescence titration. The emission maximum of the synthetase did not shift when tRNA was added. Under similar conditions ATP caused only 6%, quenching of the enzyme fluorescence from which an intrinsic association constant of 105 M−1 for about 10 moles ATP per mole subunit could be derived. On the other hand, when the enzyme was saturated with tRNA, addition of ATP increased the fluorescence intensity. Performing the titration in the reverse order, the same fluorescence end point was achieved. The addition of CTP to the preformed complex between tRNASer and the synthetase did not induce any change in the fluorescence intensity. These results are discussed with respect to the binding process and binding sites at which tRNA and ATP are attached to the synthetase and the possible involvement of the tryptophan residues.