Selective chemical modification of Escherichia coli elongation factor G: butanedione modification of an arginine essential for nucleotide binding.
Treatment of Escherichia coli elongation factor G with the arginine reagent, 2,3-butanedione, leads to the inactivation of the enzyme when performed in sodium borate buffers. The inhibition follows pseudo-first-order kinetics until 95% of the activity has been lost and further incubation results in complete inhibiton. Removal of the borate by exhaustive dialysis results in the restoration of approximately 85% of the original activity. The pH dependence of the reaction suggests that the ionization of a group in the protein with a pKa of approximately 8.8 facilitates the reaction with butanedione. A reaction order of 1.01 +/- 0.13 was calculated for the inhibition reaction, indicating that the incorporation of one butanedione per elongation factor G results in the inactivation of the enzyme. The kinetics of inhibition in the presence of GTP indicate that the elongation factor G-GTP complex is refractory to butanedione inhibiton. Elongation factor G which has been partially inactivated by butanedione has the same apparent Km for GTP as does the native enzyme. These results indicate that elongation factor G contains only one essential arginine residue which is reactive with butanedione and that this residue is located at its nucleotide binding site.
D002074:Butanones / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects*; D010445:Peptide Elongation Factors; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism
D002074:Butanones; D010445:Peptide Elongation Factors; D006160:Guanosine Triphosphate