1-N6-Etheno-ADP-ribosylation of elongation factor-2 by diphtheria toxin.
Diphtheria toxin fragment A is able to inhibit protein synthesis in the eukaryotic cell by ADP-ribosylating the diphthamide residue of elongation factor-2 (EF-2) [(1980) J. Biol. Chem. 255, 10710-10720]. The reaction requires NAD as ADP-ribose donor. This work reports on the capacity of an NAD analog, the nicotinamide 1-N6-ethenoadenine dinucleotide (epsilon NAD), to be a substrate of diphtheria toxin fragment A in the transferring reaction of the fluorescent moiety, the epsilon ADP-ribose, to the EF-2. As a consequence of the transfer of the epsilon ADP-ribosyl moiety to the EF-2, there is an increase in the emission intensity of the fluorophore and a blue shift in its emission maximum. The epsilon ADP-ribosylated EF-2, like ADP-ribosylated EF-2, retains the capacity to bind GTP and ribosome. The utility of introducing a fluorescent probe in a well defined point of the EF-2 molecule for conformational or binding studies is discussed.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D002845:Chromatography; D004167:Diphtheria Toxin / Q000378:metabolism*; D005260:Female; D006801:Humans; D007700:Kinetics; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010920:Placenta / Q000032:analysis; D011247:Pregnancy; D013050:Spectrometry, Fluorescence
D004167:Diphtheria Toxin; D009702:Nucleoside Diphosphate Sugars; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010446:Peptide Fragments; C029795:diphtheria toxin fragment A; D009243:NAD; D000246:Adenosine Diphosphate Ribose; C011038:nicotinamide 1,N(6)-ethenoadenine dinucleotide; C015195:1,N(6)-ethenoadenosine diphosphate; D000244:Adenosine Diphosphate; D010727:Phosphoric Diester Hydrolases