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The interactions among Escherichia coli elongation factor G (EF-G), guanine nucleotides, ribosomes, and fusidic acid were investigated by a number of physical techniques. Equilibrium dialysis studies demonstrated the existence of a binary EF-G-GDP complex. This complex forms with a stoichiometry of ca 1:1 and an apparent Ka of 2.5 X 10(5) M-1. While no evidence was obtained for the formation of a ribosome-GDP complex, in the presence of ribosomes, the apparent Ka for guanosine diphosphate (GDP) increased 40-fold over that for binding to EF-G alone. Although the apparent Ka increased, the stoichiometry remained ca. 1 mol of GDP/mol of EF-G. An upper limit of 1.3 X 10(7) M-1 was calculated for the Ka for binding of ribosomes to the EF-G-GDP complex. Fusidic acid had no effect on the apparent Ka's for either the EF-G-GDP or EF-G-beta, gamma-methyleneguanosine triphosphate (GMP-P(CH2)P)=ribosome complexes, but markedly increased the Ka for GDP in the EF-G-GDP-ribosome complex without altering the stoichiometry. The apparent Ka for GDP was shown to be dependent upon the fusidic acid concentration. In addition, the rate of GDP exchange into the quaternary EF-G-GDP-ribosome-fusidic acid complex was inversely related to the fusidic acid concentration. All of the data obtained in these studies suggest that the formation and dissociation of complexes involving EF-G and guanine nucleotides is ordered. GDP is the first component to bind to EF-G, followed by the ribosome, and, finally, fusidic acid. This conclusion is consistent with the kinetic mechanism for the hydrolysis of GTP by EF-G and the ribosome proposed in the preceding paper of this issue (Rohrbach and Bodley (1976b). In addition to these binding studies, guanine nucleotides have also been shown to protect EF-G against both limited trypsinolysis and chemical modification by N-ethylmaleimide. These observations offer additional evidence for the existence of a guanine nucleotide binding site on EF-G.

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.

Native elongation factor G possesses three peptide bonds or limited regions of the molecule which are especially sensitive to trypsinolysis. Cleavage at these sites occurs in sequence. Initially, the protein, a single peptide chain of 74,000 daltons, is rapidly split into a fragment of 71,000 daltons and one or more small peptides totaling 3000 daltons. This initial scission does not alter the gross three-dimentional structure of the protein, as the native and trypsin-cleaved protein have the same Kd on Sephadex gel filtration. Although cleaved, the modified elongation factor G retains full activity as measured by its ability to form complexes with the ribosome and guanine nucleotides. A single peptide bond (or region) in the 71,000-dalton fragment is then completely cleaved, yielding fragments of 47,000 and 29,000 daltons. These fragments do not remain associated under native conditions, as they are clearly resolved on gel filtration. Loss of activity is concomitant with the scission of the 71,000-dalton fragment. The third cleavage is complete under the conditions employed here and occurs in the 47,000-dalton peptide generating a fragment of 45,000 daltons and one or more small peptides totaling 2000 daltons. Prolonged treatment with higher levels of trypsin ultimately reduces the protein to small peptides but without generating further discrete fragments visible on sodium dodecyl sulfate gel electrophoresis.

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.

The interactions among Escherichia coli elongation factor G (EF-G), guanine nucleotides, ribosomes, and fusidic acid were investigated by a number of physical techniques. Equilibrium dialysis studies demonstrated the existence of a binary EF-G-GDP complex. This complex forms with a stoichiometry of ca 1:1 and an apparent Ka of 2.5 X 10(5) M-1. While no evidence was obtained for the formation of a ribosome-GDP complex, in the presence of ribosomes, the apparent Ka for guanosine diphosphate (GDP) increased 40-fold over that for binding to EF-G alone. Although the apparent Ka increased, the stoichiometry remained ca. 1 mol of GDP/mol of EF-G. An upper limit of 1.3 X 10(7) M-1 was calculated for the Ka for binding of ribosomes to the EF-G-GDP complex. Fusidic acid had no effect on the apparent Ka's for either the EF-G-GDP or EF-G-beta, gamma-methyleneguanosine triphosphate (GMP-P(CH2)P)=ribosome complexes, but markedly increased the Ka for GDP in the EF-G-GDP-ribosome complex without altering the stoichiometry. The apparent Ka for GDP was shown to be dependent upon the fusidic acid concentration. In addition, the rate of GDP exchange into the quaternary EF-G-GDP-ribosome-fusidic acid complex was inversely related to the fusidic acid concentration. All of the data obtained in these studies suggest that the formation and dissociation of complexes involving EF-G and guanine nucleotides is ordered. GDP is the first component to bind to EF-G, followed by the ribosome, and, finally, fusidic acid. This conclusion is consistent with the kinetic mechanism for the hydrolysis of GTP by EF-G and the ribosome proposed in the preceding paper of this issue (Rohrbach and Bodley (1976b). In addition to these binding studies, guanine nucleotides have also been shown to protect EF-G against both limited trypsinolysis and chemical modification by N-ethylmaleimide. These observations offer additional evidence for the existence of a guanine nucleotide binding site on EF-G.