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(ethy1eneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses of adenosine(5')tetraphospho(5')adenosine (Ap,A) and adenosine(5')triphospho(5')adenosine (Ap,A), 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 and Ap,A. It was furthermore demonstrated that the only pathway by which a synthetase-catalyzed degradation of Ap,A can occur is through the reaction between inorganic phosphate and the enzyme-bound glycyl adenylate, synthesized from Ap,A. 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 y-phosphate group from ATP to nucleoside 5'-diphosphates.The importance of the observed reaction between inorganic phosphate and enzymebound aminoacyl adenylate in relation to the remaining catalytic activities of aminoacyl-tRNA synthetases is discussed, as well as the biological significance of the reaction.

Crystal Structures and Biochemical Analyses Suggest a Unique Mechanism and Role for Human Glycyl-tRNA Synthetase in Ap4A Homeostasis

“…The tetrameric GlyRSs share low sequence similarity with the dimeric counterparts and have no conservation of insertion I (28) and therefore are not likely to use the same mechanism for Ap4A synthesis. Indeed, the formation of Ap4A by E. coli GlyRS was found to be glycine-dependent (34).…”

Diadenosine 5′,5′′′‐P1,P4‐tetraphosphate (Ap4A) controls the timing of cell division in Escherichia coli

“…It has been shown in vitro that glycyl adenylate intermediates which are bound to glycyl-tRNA synthetase (Led et al 1983), or some other aminoacyl-tRNA synthetases (Zamecnik et al 1966) donate AMP to ATP, yielding Ap4A. However, the existence of such reactions in vivo has not been demonstrated; neither aminoacyl-tRNA synthetase mutations, inhibitory treatments of aminoacylation, nor structural mutations of tRNA are known to produce Ap4A in vivo (Bochner et al 1984).…”

“…However, the existence of such reactions in vivo has not been demonstrated; neither aminoacyl-tRNA synthetase mutations, inhibitory treatments of aminoacylation, nor structural mutations of tRNA are known to produce Ap4A in vivo (Bochner et al 1984). On the contrary, glycyl-tRNA synthetase also catalyses the degradation of Ap4A to ADP in the presence of PPi (Led et al 1983). Thus the increase in Ap4A concentration by the cfcA11 mutation is caused either by an enhanced synthesis of Ap4A, or by the inhibition of degradation.…”

The B Form of Dihydroorotate Dehydrogenase from Lactococcus lactis Consists of Two Different Subunits, Encoded by the pyrDb and pyrK Genes, and Contains FMN, FAD, and [FeS] Redox Centers

“…The Sequenase 2.0 kit was from U.S. Biochemical Corp. Diethylaminoethyl-cellulose (DE52) was from Whatman BioSystems Ltd. (Maidstone, United Kingdom), hydroxylapatite (Bio-Gel) was from Bio-Rad, the dye-liganded Matrex Red A was from Amicon, and the Superose 12 column was from Pharmacia (Uppsala, Sweden). Blue dextran-Sepharose was prepared as described previously (Led et al, 1983). Molecular weight marker proteins were bought from Bio-Rad or Pharmacia.…”