PMID:11744709
| Citation |
Wieden, HJ, Gromadski, K, Rodnin, D and Rodnina, MV (2002) Mechanism of elongation factor (EF)-Ts-catalyzed nucleotide exchange in EF-Tu. Contribution of contacts at the guanine base. J. Biol. Chem. 277:6032-6 |
|---|---|
| Abstract |
Nucleotide exchange in elongation factor Tu (EF-Tu) is catalyzed by elongation factor Ts (EF-Ts). Similarly to other GTP-binding proteins, the structural changes in the P loop and the Mg(2+) binding site are known to be important for nucleotide release from EF-Tu. In the present paper, we determine the contribution of the contacts between helix D of EF-Tu at the base side of the nucleotide and the N-terminal domain of EF-Ts to the catalysis. The rate constants of the multistep reaction between Escherichia coli EF-Tu, EF-Ts, and GDP were determined by stopped-flow kinetic analysis monitoring the fluorescence of either Trp-184 in EF-Tu or mant-GDP. Mutational analysis shows that contacts between helix D of EF-Tu and the N-terminal domain of EF-Ts are important for both complex formation and the acceleration of GDP dissociation. The kinetic results suggest that the initial contact of EF-Ts with helix D of EF-Tu weakens binding interactions around the guanine base, whereas contacts of EF-Ts with the phosphate binding side that promotes the release of the phosphate moiety of GDP appear to take place later. This "base-side-first" mechanism of guanine nucleotide release resembles that found for Ran x RCC1 and differs from mechanisms described for other GTPase x GEF complexes where interactions at the phosphate side of the nucleotide are released first. |
| Links |
PubMed Online version:10.1074/jbc.M110888200 |
| Keywords |
Binding Sites; Escherichia coli/genetics; Escherichia coli/metabolism; Guanosine Diphosphate/metabolism; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Peptide Elongation Factor Tu/chemistry; Peptide Elongation Factor Tu/metabolism; Peptide Elongation Factors/chemistry; Peptide Elongation Factors/metabolism; Protein Conformation; Protein Structure, Secondary; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism |
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