Mechanism of EF-Ts-catalyzed guanine nucleotide exchange in EF-Tu: contribution of interactions mediated by helix B of EF-Tu.
Elongation factor Tu (EF-Tu) belongs to the family of GTP-binding proteins and requires elongation factor Ts (EF-Ts) for nucleotide exchange. Crystal structures suggested that one of the salient features in the EF-Tu x EF-Ts complex is a conformation change in the switch II region of EF-Tu that is initiated by intrusion of Phe81 of EF-Ts between His84 and His118 of EF-Tu and may result in a destabilization of Mg2+ coordination and guanine nucleotide release. In the present paper, the contribution of His84 to nucleotide release was studied by pre-steady-state kinetic analysis of nucleotide exchange in mutant EF-Tu in which His84 was replaced by Ala. Both intrinsic and EF-Ts-catalyzed nucleotide release was affected by the mutation, resulting in a 10-fold faster spontaneous GDP release and a 4-fold faster EF-Ts-catalyzed release of GTP and GDP. Removal of Mg2+ from the EF-Tu x EF-Ts complex increased the rate constant of GDP release 2-fold, suggesting a small contribution to nucleotide exchange. Together with published data on the effects of mutations interfering with other putative interactions between EF-Tu and EF-Ts, the results suggest that each of the contacts in the EF-Tu x EF-Ts complex alone contributes moderately to nucleotide destabilization, but together they act synergistically to bring about the overall 60,000-fold acceleration of nucleotide exchange in EF-Tu by EF-Ts.
Schümmer
Tobias
T
Institute of Physical Biochemistry, University of Witten/Herdecke, D-58448 Witten, Germany.
Gromadski
Kirill B
KB
Rodnina
Marina V
MV
D002384:Catalysis; D006150:Guanine Nucleotides / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011487:Protein Conformation
D006150:Guanine Nucleotides; D010445:Peptide Elongation Factors; C023512:elongation factor Ts; D010444:Peptide Elongation Factor Tu