title: Localization of the active site of diphtheria toxin.
abstract: Information about the location of the active site of diphtheria toxin was derived from proteolysis studies and an analysis of its sequence. It was found that a specific trypsin cleavage within whole diphtheria toxin occurs at Lys-39. Therefore, Lys-39 appears to be a surface residue. Furthermore, protection from proteolysis could be obtained upon binding of either the substrate beta-nicotinamide adenine dinucleotide (oxidized form) (NAD+) or a competing ligand, adenylyl(3'-5')uridine 3'-phosphate (ApUp). The protection by ApUp, which binds to the toxin very tightly, required only stoichiometric levels. The most likely explanation of these results is that both NAD+ binding and ApUp binding block trypsin either through a steric mechanism or through a local conformational change, suggesting Lys-39 may be near the active site. Further evidence supporting this conclusion comes from comparison of the previously determined sequences of diphtheria toxin and of Pseudomonas exotoxin A, a protein that catalyzes an identical reaction. We find a significant degree of homology between the N-terminal halves of the catalytic domains of these two proteins, which apparently represents active-site residues, and that Lys-39 is in the center of the homologous sequence. Furthermore, the location of the amino acid that is the homologue of Lys-39 within the crystal structure of Pseudomonas exotoxin A is also in agreement with a location in or near the active site. Other unusual features in the sequences of diphtheria toxin and Pseudomonas exotoxin A are also described, and on the basis of the experiments presented, a possible function for ApUp is considered.
authors.affiliation: Department of Biochemistry, State University of New York, Stony Brook 11794-5215.
authors.forename: J M
authors.initials: JM
authors.lastname: Zhao
authors.forename: E
authors.initials: E
authors.lastname: London
mesh_terms: D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001427:Bacterial Toxins; D001665:Binding Sites; D004167:Diphtheria Toxin / Q000378:metabolism*; D005098:Exotoxins; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D014357:Trypsin; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D004167:Diphtheria Toxin; D005098:Exotoxins; D009243:NAD; D009843:Oligoribonucleotides; D010446:Peptide Fragments; D014357:Trypsin; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C043846:adenylyl-(3'-5')-uridine 3'-monophosphate