The catalytic mechanism of the glutamyl-tRNA synthetase from Escherichia coli. Detection of an intermediate complex in which glutamate is activated.
Up to now it was not possible to isolate an enzyme . adenylate complex after mixing the glutamyl-tRNA synthetase from Escherichia coli with ATP, MgCl2, and glutamate. This enzyme catalyzes an AMP-dependent and PPi-independent deacylation of Glu-tRNAGlu. The labeled glutamate which disappears from Glu-tRNAGlu in the presence of AMP remains linked to the enzyme in a complex isolated by filtration on nitrocellulose discs. The addition of tRNAGlu to this reaction mixture at the deacylation plateau gives rise to a synthesis of Glu-tRNAGlu, via an ATP-independent reaction. These results indicate the existence of the following equilibrated reaction catalyzed by the glutamyl-tRNA synthetase E + Glu-tRNAGlu + AMP in equilibrium E . AMP approximately Glu + tRNAGlu. This transfer of glutamate from an activated complex to tRNAGlu indicates that the formation of glutamyl-tRNA is catalyzed via a two-step reaction mechanism. The AMP-dependent and PPi-independent deacylation of Glu-tRNAGlu is the rate-limiting step of the reverse of the AMP- and PPi-dependent deacylation.
D000249:Adenosine Monophosphate; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005975:Glutamate-tRNA Ligase / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl; D046249:Transfer RNA Aminoacylation
D005971:Glutamates; D012346:RNA, Transfer, Amino Acyl; D000249:Adenosine Monophosphate; D000604:Amino Acyl-tRNA Synthetases; D005975:Glutamate-tRNA Ligase