A point mutation within each of two ATP-binding motifs inactivates the functions of elongation factor 3

We have investigated how point mutations in the two ATP-binding motifs (G⁴⁶³PNGCGK⁴⁶⁹ST and G⁷⁰¹PNGAGK⁷⁰⁷ST) of elongation factor 3 (EF-3) affect ribosome-activated ATPase activity of EF-3, polyphenylalanine synthesis, and growth of Saccharomyces cerevisiae. The point mutation impaired the ribosome-activated ATPase activity of EF-3, when glycine^{463 and 701} and lysine^{469 and 707} were replaced with valine and arginine, respectively. Thus, each glycine and lysine residue in both ATP-binding motifs is indispensable for EF-3's binding with ATP and the ensuing generation of ribosome-activated ATPase activity. Additionally, the mutant EF-3s did not catalyze polyphenylalanine synthesis in vitro when each glycine^{463 and 701} was replaced with valine. The mutant EF-3s did not support cell growth in TEF3-disrupted S. cerevisiae, when each lysine^{469 and 707} and glycine⁴⁶³ was replaced with arginine and valine, respectively. Thus, each of the two ATP-binding motifs of EF-3 is indispensable for the ribosome-activated ATPase activity of EF-3, which is required for protein synthesis and cell growth in S. cerevisiae.