Kinetic Mechanism of Elongation Factor Ts-Catalyzed Nucleotide Exchange in Elongation Factor Tu

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Abstract
The interaction of Escherichia coli elongation factor Tu (EF-Tu) with elongation factor Ts (EF-Ts) and guanine nucleotides was studied by the stopped-flow technique, monitoring the fluorescence of tryptophan 184 in EF-Tu or of the mant group attached to the guanine nucleotide. Rate constants of all association and dissociation reactions among EF-Tu, EF-Ts, GDP, and GTP were determined. EF-Ts enhances the dissociation of GDP and GTP from EF-Tu by factors of 6 × 104 and 3 × 103, respectively. The loss of Mg2+ alone, without EF-Ts, accounts for a 150−300-fold acceleration of GDP dissociation from EF-Tu·GDP, suggesting that the disruption of the Mg2+ binding site alone does not explain the EF-Ts effect. Dissociation of EF-Ts from the ternary complexes with EF-Tu and GDP/GTP is 103−104 times faster than from the binary complex EF-Tu·EF-Ts, indicating different structures and/or interactions of the factors in the binary and ternary complexes. Rate constants of EF-Ts binding to EF-Tu in the free or nucleotide-bound form or of GDP/GTP binding to the EF-Tu·EF-Ts complex range from 0.6 × 107 to 6 × 107 M-1 s-1. At in vivo concentrations of nucleotides and factors, the overall exchange rate, as calculated from the elemental rate constants, is 30 s-1, which is compatible with the rate of protein synthesis in the cell.