title: Diphtheria toxin. Effect of substituting aspartic acid for glutamic acid 148 on ADP-ribosyltransferase activity.
abstract: Photoaffinity labeling experiments with diphtheria toxin fragment A have implicated glutamic acid 148 as a constituent of the NAD binding site. To evaluate the role of this residue in ADP-ribosylation of elongation factor 2, we replaced it with aspartic acid by in vitro mutagenesis of a toxin gene fragment cloned in Escherichia coli. Fragment A containing aspartic acid at position 148 had less than 0.6% the ADP-ribosylation activity of wild-type fragment A. The mutation produced no change in sensitivity of fragment A to trypsin and little, if any, reduction in affinity of fragment A for NAD. These results indicate that glutamic acid 148 is essential for the ADP-ribosylation of elongation factor 2 and are consistent with other data suggesting that this residue may be at or near the catalytic center of the toxin.
authors.forename: R K
authors.initials: RK
authors.lastname: Tweten
authors.forename: J T
authors.initials: JT
authors.lastname: Barbieri
authors.forename: R J
authors.initials: RJ
authors.lastname: Collier
mesh_terms: D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003353:Corynebacterium diphtheriae / Q000235:genetics; D004167:Diphtheria Toxin / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D005971:Glutamates; D018698:Glutamic Acid; D009154:Mutation; D009243:NAD / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases; D011485:Protein Binding
chemical_list: D004167:Diphtheria Toxin; D005971:Glutamates; D009243:NAD; D001224:Aspartic Acid; D018698:Glutamic Acid; D011065:Poly(ADP-ribose) Polymerases; D009713:Nucleotidyltransferases