Kinetic mechanism of elongation factor Ts-catalyzed nucleotide exchange in elongation factor Tu.
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 Mg(2+) alone, without EF-Ts, accounts for a 150-300-fold acceleration of GDP dissociation from EF-Tu.GDP, suggesting that the disruption of the Mg(2+) 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.
Gromadski
Kirill B
KB
Institute of Physical Biochemistry, University of Witten/Herdecke, D-58448 Witten, Germany.
Wieden
Hans-Joachim
HJ
Rodnina
Marina V
MV
D001426:Bacterial Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D006150:Guanine Nucleotides / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008433:Mathematics; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D006150:Guanine Nucleotides; D010445:Peptide Elongation Factors; C023512:elongation factor Ts; D010444:Peptide Elongation Factor Tu; D008274:Magnesium