The yeast peptide elongation factor 3 (EF-3) carries an active site for ATP hydrolysis which can interact with various nucleoside triphosphates in the absence of ribosomes.

ATP (GTP) hydrolysis was clearly demonstrated by using at most 16 pmol of yeast peptide elongation factor 3 (EF-3) in the absence of ribosomes. However, the highly active yeast ribosomes (up to 48 pmol) displayed virtually no ATPase (or GTPase) activity in the absence of EF-3. Several lines of evidence indicated that both the catalytic and binding sites of the ATPase reside in the elongation factor itself, not on the ribosomes. The patterns of protection by various nucleoside triphosphates against tryptic digestion of EF-3, reflecting the wide substrate specificity of the ATPase, confirmed that the active center of the endogenous ATPase is located on the factor itself and not on contaminants. The intrinsic activity was stimulated up to two orders of magnitude by the presence of the yeast ribosomes fully active in polyphenylalanine synthesis. The activation was achieved by enhancing the catalytic activity (kcat) to a much greater extent than the binding affinity (Km). On the other hand, the ribosome-activated ATPase activity was revealed to inherit its wide substrate specificity from the intrinsic property of EF-3, which shows an affinity to various XTPs, including pyrimidine- and purine-nucleoside triphosphates, irrespective of 2'-hydroxylation of the sugar moiety. From experiments on protection against tryptic digestion, we determined that intricate conformational changes of the factor molecule occur upon interaction with the substrate XTP and ribosomes.