PMID: 764868Feb 24, 1976

Mechanism of aminoacylation of tRNA. Proof of the aminoacyl adenylate pathway for the isoleucyl- and tyrosyl-tRNA synthetases from Escherichia coli K12

A R Fersht, M M Kaethner


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 overa...Continue Reading


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Related Concepts

Amino Acid Activation, Translational
Alkalescens-Dispar Group
Transfer RNA
Amino Acyl-tRNA Synthetases
Isoleucine-tRNA Ligase
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