Mechanism of aminoacylation of tRNA. Proof of the aminoacyl adenylate pathway for the isoleucyl- and tyrosyl-tRNA synthetases from Escherichia coli K12.
The following observations show that the formation of isoleucyl-tRNA catalyzed by the isoleucyl-tRNA synthetase from Escherichia coli K12 involves the initial rapid formation of an isoleucyl adenylate complex followed by the slow, rate-determining, transfer of the isoleucyl moiety to tRNA. (1) The rate constant for the transfer of [14C]Ile from the E-[14C]Ile approximately AMP complex to tRNA is the same as the turnover number for the steady-state isoleucylation of tRNA at pH 7.78 (1.5 s-1) and pH 5.87 (0.34 s-1). (2) On mixing a solution of isoleucyl-tRNA synthetase and tRNA with [14C]Ile and ATP the steady-state rate of isoleucylation is attained in the first turnover of the enzyme, with little or no "burst" or charging that would indicate a slow step after the transfer step. (3) The pyrophosphate exchange reaction in the presence of tRNA is 40 times faster than the overall rate of isoleucylation of tRNA. (4) Similarly, rapid quenching experiments indicate that isoleucyl adenylate is formed prior to the transfer step. The possibility that isoleucyl adenylate formation is a parallel reaction caused by a second active site on the enzyme is ruled out both by the stoichiometry in this rapid quenching experiment and also the overall stoichiometry of isoleucyl-tRNA formation. At saturating reagent concentrations the major species in solution is the E-tRNA-Ile approximately AMP complex. Similar observations are found for the tyrosyl-tRNA systhetase except that at saturating reagent concentrations the rate constants for both tyrosyl adenylate formation and transfer are similar so that both processes contribute to the rate-determining step.
D000249:Adenosine Monophosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007532:Isoleucine / Q000378:metabolism; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D012329:RNA, Bacterial / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D046249:Transfer RNA Aminoacylation; D014443:Tyrosine / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000378:metabolism*
D000596:Amino Acids; D012329:RNA, Bacterial; D007532:Isoleucine; D000249:Adenosine Monophosphate; D014443:Tyrosine; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase; D007533:Isoleucine-tRNA Ligase