Photoaffinity labeling of chloroplastic and cytoplasmic leucyl-tRNA synthetases of Euglena gracilis by the gamma-(p-azidoanilidate) of ATP.
The photoreactive gamma-(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 gamma-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 concentration-dependent and time-dependent manner indicative of photoaffinity labeling. Both ATP and leucine, but not tRNA, protect the enzymes against ultraviolet-induced inactivation by AzAnATP. Comparative kinetic characterization of the inactivation process reveals differences in the active centers of the two intracellular isoenzymes.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000345:Affinity Labels; D000604:Amino Acyl-tRNA Synthetases / Q000032:analysis*; D001386:Azides; D001665:Binding Sites / Q000187:drug effects; D002736:Chloroplasts / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology*; D005056:Euglena gracilis / Q000201:enzymology*; D007935:Leucine-tRNA Ligase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000528:radiation effects; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; C024294:ATP gamma-p-azidoanilide; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; D007935:Leucine-tRNA Ligase