Yeast phenylalanyl-tRNA synthetase. Affinity and photoaffinity labelling of the stereospecific binding sites.

The localization of the binding sites of the different ligands on the constitutive subunits of yeast phenylalanyl-tRNA synthetase was undertaken using a large variety of affinity and photoaffinity labelling techniques. The RNAPhe was cross-linked to the enzyme by non-specific ultraviolet irradiation at 248 nm, specific irradiation in the wye base absorption band (315 nm), irradiation at 335 nm, in the absorption band of 4-thiouridine (S4U) residues introduced in the tRNA molecule, or by Schiff's base formation between periodate-oxidized tRNAPhe (tRNAPheox) and the protein. ATP was specifically incorporated in its binding site upon photosensitized irradiation. The amino acid could be linked to the enzyme upon ultraviolet irradiation, either in the free state, engaged in the adenylate or bound to the tRNA. The tRNA, the ATP molecule and the amino acid linked to the tRNA were found to interact exclusively with the beta subunit (Mr 63000). The phenylalanine residue, either free or joined to the adenylate, could be cross-linked with equal efficiency to eigher type of subunit, suggesting that the amino acid binding site is located in a contact area between the two subunits. The Schiff's base formation between tRNAPheox and the enzyme shows the existence of a lysyl group close to the binding site for the 3'-terminal adenosine of tRNA. This result was confirmed by the study of the inhibition of yeast phenylalanyl-tRNA synthetase with pyridoxal phosphate and the 2',3'-dialdehyde derivative of ATP, oATP.