Kinetic determination of the effects of ADP-ribosylation on the interaction of eukaryotic elongation factor 2 with ribosomes.
The effect of ADP-ribosylation on the function of eukaryotic elongation factor 2 (EF-2) was investigated by kinetic analysis of the EF-2-catalyzed hydrolysis of GTP in the presence of ribosomes and by direct determination of the affinity of the modified factor for the ribosome. Under conditions where the concentration of EF-2 was rate-limiting, the ADP-ribosylation reduced the maximum rate of GTP hydrolysis and the second order rate constant Kcat/Km by approximately 50%. A similar decrease in Kcat and Kcat/Km was observed when the concentration of ribosomes were kept rate-limiting. The affinity of EF-2 for the pretranslocation type of ribosomes was reduced by 2 orders of magnitude after ADP-ribosylation. No effect was observed in the interaction with the post-translocation type of ribosomes, the ribosomal conformation responsible for activation of the EF-2-dependent GTPase. We conclude that the ADP-ribosylation affects both the association of the modified factor with pretranslocation ribosomes and the hydrolytic capacity of the factor.
Nygård
O
O
Department of Cell Biology, Wenner-Gren Institute, University of Stockholm, Sweden.
Nilsson
L
L
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism; D004167:Diphtheria Toxin / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D009243:NAD / Q000378:metabolism*; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D012276:Ricin / Q000494:pharmacology
D004167:Diphtheria Toxin; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010750:Phosphoproteins; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D006160:Guanosine Triphosphate; D012276:Ricin