Photoaffinity Leabeling of Chloroplastic and Cytoplasimic Leycly‐rRNA Synthetases of Euglena gracilis, by the γ‐(p‐Azidoanilidae) of ATP

Abstract

The photoreactive y-( p-azidoanilidate) analog of ATP, AzAnATP, was used to affinity-label the chloroplastic and cytoplasmic leucyl-tRNA synthetases of Euglena gracilis. The analog is able to replace the substrate ATP in the tRNA leucylation reaction catalyzed by both enzymes. In the presence of ATP, it is a competitive inhibitor against ATP as well as leucine for the two isoenzymes, as is also shown for the photoinactive y-anilidate analog of ATP, AnATP, which does not serve as substrate in the enzyme reaction.During ultraviolet irradiation, the enzymes are irreversibly inactivated by AzAnATP in a concentrationdependent and time-dependent manner indicative of photoaffinity labeling. Both ATP and leucine, but not tRNA, protect the enzymes against ultraviolet-induced inactivation by AzAn ATP. Comparative kinetic characterization of the inactivation process reveals differences in the active centers of the two intracellular isoenzymes.