The mechanism of aminoacylation of transfer ribonucleic acid. Reactivity of enzyme-bound isoleucyl adenylate.
Isoleucyl adenylate bound to isoleucine:tRNA ligase of Escherichia coli (EC 6.1.1.5; isoleucyl-tRNA synthetase) transfers the isoleucine moiety to tRNA-Ile-E. coli with a half-time of about 35 s at 0 degrees and pH 7.6 in the presence of spermine or Mg2+. If a limited amount of tRNA-Ile is supplied to a mixture of free enzyme and enzyme-bound [14c]isoleucyl adenylate in a medium containing spermine, ATP, and [3H]isoleucine, almost none of the resultant isoleucyl tRNA is derived from preformed enzyme-bound [14C]isoleucyl adenylate. Almost all of the isoleucyl tRNA formed results directly from reaction of free enzyme, ATP, and isoleucine with tRNA. Similar but less clearcut results are obtained when Mg2+ is substituted for spermine. We conclude that isoleucyl adenylate bound to isoleucine:tRNA ligase is not a significant intermediate in the synthesis of isoleucyl tRNA under these conditions.
D000249:Adenosine Monophosphate; D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002845:Chromatography; D004926:Escherichia coli / Q000201:enzymology*; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011485:Protein Binding; D013096:Spermine / Q000494:pharmacology; D046249:Transfer RNA Aminoacylation
D013096:Spermine; D000249:Adenosine Monophosphate; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase; D008274:Magnesium