Binding of periodate-oxidized guanine nucleotides to eukaryotic elongation factor 2

Abstract

Periodate-oxidized guanine nucleotides (GTP ox and GDP ox) were shown to bind stoichiometrically to rat liver elongation factor 2 (EF-2). This binding was quantitatively inhibited in the presence of GTP. After binding, oxidized nucleotides remained on EF-2 despite extensive dialysis. They exchanged, however, with free quanine nucleotides in the course of prolonged (> 1 h) incubations. The prior reduction EF-2 · GTP ox with NaBH 4 abolished, to a large extent, this slow exchange. Thus, a Schiff's base was implicated to be formed between EF-2 and oxidized guanine nucleotides. Mg 2+ increased the GTP ox concentration necessary for a stoichiometric binding to EF-2. EF-2-oxidized nucleotide conjugates bound in the presence of ribosomes a second molecule of GTP (or GTP ox). GTP ox bound to EF-2 in the presence of ribosomes appeared to exchange readily with free GTP. Moreover, GTP ox proved to be active as substrate in EF-2 and ribosome-dependent GTPase reaction: K m values found for GTP ox and GTP were 7.7 and 3.4 μM, respectively. The binding of GTP ox to EF-2 inhibited only partially the subsequent ribosome-dependent GTP binding, and GTPase reaction or polyphenylalanine (polyPhe) synthesis. On the other hand, the binding of Guo PP[CH 2] P ox to EF-2 inhibited all of these reactions strongly. The nature of the binding site involved in the direct interactions of EF-2 with guanine nucleotides is discussed in the light of these results.