Aminoacyl Transfer RNA Formation

VII. Lack of Correlation between Aminoacylation and PP_i-ATP Exchange Catalyzed by Isoleucyl-tRNA Synthetase of Escherichia coli in the Presence of Various Divalent Cations

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Isoleucyl-tRNA formation and isoleucine-dependent PP₁-ATP exchange catalyzed by purified isoleucyl-tRNA synthetase [EC 6.1.1.5] of Escherichia coli were studied in the presence of various amounts of either Mg²⁺, Ca²⁺, Fe²⁺, Ni²⁺, or Cu²⁺. In the presence of Mg²⁺, isoleucine-dependent PP₁-ATP exchange was observed in parallel with isoleucyl-tRNA formation, while in the presence of Ca²⁺, isoleucyl-tRNA for-mation was observed without isoleucine-dependent PP, -ATP exchange. Moreover, isoleucine-dependent PP_i-ATP exchange was much more in the presence of Fe²⁺ than in the presence of Mg²⁺, while little isoleucyl-tRNA was formed in the presence of Fe²⁺. In the presence of Ni²⁺ or Cu²⁺, neither reaction was observed.
These data, indicating that formation of an isoleucyl-AMP-enzyme complex is not a necessary step in isoleucyl-tRNA formation, support the existence of a concerted mechanism of isoleucyl-tRNA formation in E. coli.