Properties and Function of the Sulfhydryl Group in the Polypeptide Chain Elongation Factor G from E. coli

Abstract

The properties and function of sulfhydryl groups in the polypeptide chain elongation factor G (EF-G) have been studied by titration with p-chloromercuri[14C]benzoate or 5,5′-dithiobis(2-nitrobenzoic acid), and by assessing the inhibitory effect of N-ethylmaleimide on its activity. EF-G contained three sulfhydryl groups per mole of protein, of which only one was reactive under native conditions. The other two were nonreactive and could be titrated only after complete denaturation of the protein. The activity to catalyze the uncoupled GTPase reaction as well as the ability to form a ternary complex involving EF-G, guanine nucleotides, and ribosomes was completely inactivated by treatment of EF-G with N-ethylmaleimide. The reactive sulfhydryl group was not required for interaction with guanine nucleotides, but was essential for binding to ribosomes. The binary complex involving EF-G and GDP could be isolated after treatment of EF-G with sulfhydryl reagents. The sulfhydryl group of EF-G was not protected by guanine nucleotides alone, but a marked protection was afforded by addition of ribosomes and GMP-P(CH2)P. The reactivity of the sulfhydryl group of EF-G was modulated by interaction with guanine nucleotides. The kinetic studies indicated that reactivity toward N-(1-anilinonaphthyl-4)-maleimide was reduced by GDP and, to a lesser extent, by GTP. These results, together with the previous report on the spin-label probes (Arai, N., Arai, K., Maeda, T., Ohnishi, S., and Kaziro, Y. (1976) J. Biochern. 80, 1057–1065) indicate that (1) the reactive sulfhydryl group of EF-G is required for interaction with ribosomes, and (2) its reactivity is modulated by guanine nucleotide-induced conformational transitions occurring near the reactive protein sulfhydryl.