title: Phosphorolytic activity of Escherichia coli glycyl-tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P-NMR spectroscopy and thin-layer chromatography.
abstract: The catalytic activity of highly purified Escherichia coli glycyl-tRNA synthetase has been studied by 31P-NMR spectroscopy and thin-layer chromatography on poly(ethyleneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses of adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')triphospho(5')adenosine (Ap3A), also catalyzes the formation of ADP from inorganic phosphate and the enzyme-bound glycyl adenylate. Accordingly it was shown that E. coli glycyl-tRNA synthetase, in the presence of inorganic phosphate, glycine, and Mg2+ ions, catalyzes the synthesis of ADP from three different substrates which all lead to enzyme-bound glycyl adenylate, that is, ATP, adenosine 5'-[beta, gamma-methylene]triphosphate and Ap4A. It was furthermore demonstrated that the only pathway by which a synthetase-catalyzed degradation of Ap4A can occur is through the reaction between inorganic phosphate and the enzyme-bound glycyl adenylate, synthesized from Ap4A. Likewise a 20-fold increase of the phosphorolytic activity of the investigated synthetase was observed when Mg2+ was replaced by Mn2+. Besides establishing the phosphorolytic activity of the applied enzyme, the study also showed that the preparation catalyzes a glycine-independent transfer of the gamma-phosphate group from ATP to nucleoside 5'-diphosphates. The importance of the observed reaction between inorganic phosphate and enzyme-bound aminoacyl adenylate in relation to the remaining catalytic activities of aminoacyl-tRNA synthetases is discussed, as well as the biological significance of the reaction.
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000502:physiology*; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000201:enzymology*; D006032:Glycine-tRNA Ligase / Q000502:physiology*; D009682:Magnetic Resonance Spectroscopy; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding
chemical_list: D000227:Adenine Nucleotides; D009701:Nucleoside-Diphosphate Kinase; D000604:Amino Acyl-tRNA Synthetases; D006032:Glycine-tRNA Ligase