Characterization of a full-length, active-site mutant of diphtheria toxin.
A full-length recombinant mutant of diphtheria toxin containing serine in place of a crucial active-site glutamate has been purified and characterized. The serine substitution caused a minor structural alteration in the toxin as measured by trypsinolysis. ADP-ribosyltransferase activity and cytotoxicity of the mutant were both decreased by approximately 500-fold. A similar reduction in cytotoxicity was found when the enzymic fragments of both the wild-type and mutant toxins were introduced into the cytosol of fibroblasts by osmotically lysing pinosomes. The mutation did not alter the binding of the toxin to cell surface receptors and had no apparent effect on membrane translocation. The results suggest that the decreased cytotoxicity of the mutant is solely due to the reduced ADP-ribosyltransferase activity.
O'Keefe
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Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005971:Glutamates / Q000737:chemistry; D018698:Glutamic Acid; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D016296:Mutagenesis; D009997:Osmotic Pressure; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012694:Serine / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
D004167:Diphtheria Toxin; D005971:Glutamates; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011500:Protein Synthesis Inhibitors; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins; D018698:Glutamic Acid; D012694:Serine; D011065:Poly(ADP-ribose) Polymerases; D014357:Trypsin