Kinetic mechanism of elongation factor Ts-catalyzed nucleotide exchange in elongation factor Tu

被引:98
作者
Gromadski, KB [1 ]
Wieden, HJ [1 ]
Rodnina, MV [1 ]
机构
[1] Univ Witten Herdecke, Inst Phys Biochem, D-58448 Witten, Germany
关键词
D O I
10.1021/bi015712w
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 x 10(4) and 3 x 10(3), 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 10(3)-10(4) 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 x 10(7) to 6 x 10(7) 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.
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页码:162 / 169
页数:8
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