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.