The histidine residue of codon 715 is essential for function of elongation factor 2

Abstract

Several mutant cDNAs of elongation factor 2 (EF‐2) were constructed by site‐directed mutagenesis and their products expressed in mouse cells were investigated. Amino acid substitution for the histidine residue of codon 715, which is modified post‐translationally to diphthamide, resulted in non‐functional EF‐2 and this substitution did not render EF‐2 resistant to Pseudomonas aeruginosa exotoxin A, which inactivates EF‐2 transferring ADP‐ribose to the diphthamide residue. These non‐functional EF‐2s with replacements of the histidine‐715 residue showed various extents of inhibition of protein synthesis by competing with functional EF‐2 in vivo. These results suggest that histidine‐715 is essential for the translocase activity of EF‐2 and that the region around diphthamide functions in recognition of, and/or binding to ribosomes. Substitution of proline for the alanine‐713 residue and substitution of glutamine for the glycine‐717 residue converted EF‐2 to partially toxin‐resistant forms. Two‐dimensional gel analysis with fragment A of diphtheria toxin of these toxin‐resistant EF‐2s revealed that their ADP‐ribosylations by toxin were much less than that of wild‐type EF‐2. Copyright © 1989, Wiley Blackwell. All rights reserved