Localization of the active site of diphtheria toxin.
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.
Zhao
J M
JM
Department of Biochemistry, State University of New York, Stony Brook 11794-5215.
London
E
E
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
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