Phenylalanyl-tRNA synthetase from E. coli MRE-600: analysis of the active site distribution on the enzyme subunits by affinity labelling.
Affinity labelling has been employed to localize the substrate-binding sites on the enzyme subunits of phenylalanyl-tRNA synthetase (L-phenylalanine:tRNAPhe-ligase, EC 6.1.1.20) of Escherichia coli MRE-600 (alpha 2 beta 2-type). N-Chlorambucilylphenylalanyl-tRNA, N-bromoacetylphenylalanyl-tRNA, tRNAPhe containing an azido group at the eighth position of the molecule (S4U), tRNAPhe containing azido groups at different points of the molecule, p-azidoanilidate of phenylalanine, adenosine 5'-trimethaphosphate and N-bromoacetyl-L-phenylalaninyladenylate were used in experiments. It has been shown that tRNA-binding sites are formed on heavy beta-subunits of the enzyme. Phenylalanyl-tRNA is also localized on beta-subunits, while the aminoacyl moiety of aminoacyl-tRNA is localized near the contact region of subunits. The phenylalanine-binding site is located on light alpha-subunits of the enzyme. Adenosine 5'-trimethaphosphate and the analogue of phenylalanyladenylate modify both types of enzyme subunits. In our opinion, the catalytic center of tRNA aminoacylation is formed in the contact region of subunits, and the aminoacyl moiety is transferred into tRNA (from the alpha- into beta-subunit in the region of their contact).
D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D046911:Macromolecular Substances; D008958:Models, Molecular; D010649:Phenylalanine / Q000378:metabolism; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism*
D000345:Affinity Labels; D046911:Macromolecular Substances; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase