A kinetic analysis of the interaction of elongation factor Tu with guanosine nucleotides and elongation factor Ts.
The interaction of elongation factor Tu (EF-Tu) and elongation factor Ts (EF-Ts) from Escherichia coli has been investigated by kinetic methods. It was found that EF-Ts purified on an EF-Tu affinity column contained a transphosphorylase activity which could transfer the gamma-phosphate of GTP to [3H]GDP. However, this activity showed different sensitivities to heat and N-ethylmaleimide compared to the EF-Ts activity. Using the chromophoric GDP analogue, 2-amino-6-mercaptopurine riboside 5'-diphosphate (thioGDP), spectrophotometric titrations and stopped-flow experiments enabled the interaction of EF-Tu X thioGDP with EF-Ts and of EF-Tu X EF-Ts with thioGDP to be investigated. The results were analyzed according to the scheme of Chau et al. (Chau, V., Romero, G., and Biltonen, R.L. (1981) J. Biol. Chem. 256, 5591-5596). (Formula: see text) Values for the rate constants obtained were k1 greater than or equal to 2 X 10(8) M-1 s-1, k-1 greater than or equal to 2600 s-1, k2 = 500 s-1, and k-2 = 4 X 10(5) M-1 s-1. The most notable feature of these results is that EF-Ts binds to EF-Tu X thioGDP at a rate approaching that expected for a diffusion-controlled reaction whereas thioGDP binds to EF-Tu X EF-Ts several orders of magnitude more slowly than this. The relevance of these results to the interactions involving GDP is discussed.
D004926:Escherichia coli / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D010444:Peptide Elongation Factor Tu; D010445:Peptide Elongation Factors / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism; D014316:Tritium
D006150:Guanine Nucleotides; D010445:Peptide Elongation Factors; D013873:Thionucleotides; C023512:elongation factor Ts; D014316:Tritium; D006153:Guanosine Diphosphate; C032829:6-thioguanosine 5'-diphosphate; C032827:6-thioguanosine 5'-triphosphate; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu