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

Abstract

The photoreactive-δ(p-azidoanilidate) analog of ATP, AzAn ATP, was used to affinity-label the chlorophlastic an cytoplasmic leucyl-tRNA synthetases of Euglena gracilis. The analog is abvle to replacxe the substrate ATP is in the tRNA leucylation reaction catalyzed by both enzymes. In the presence of ATP, it is competitive inhibitor against ATP as well a sleucine for the two isoenzymes, as is also shown for the photoiactive- δ-anilidate analog of ATP, AnATP, which does not serve as subtrate in the enzyme reaction. During ultraviolet irradiaiton, the enzymes are irrevesibly inactivated by AzAnATP in a concenytration-dependent and time-dependent manner indicative of phtoaffinity labelling. Both ATP and leucine, but not tRNA, protect the enzymes against ultraviolet-induced inactivation by AzAn ATP. Comparative kinetic characterization of the inactivation process differences in the active centers of two intracelluar isoenzymes.