Diphtheria Active site

Wilson BA, Collier RJ (1992) Diphtheria toxin and Pseudomonas aeruginosa exotoxin A Active-site structure and enzymatic mechanism. In Curr. Top. Microbiol. Immunol. 175 27-41. 

Fig. 1. The active sites of PT SI and of the A subunits of diphtheria toxin and Escherichia coii heat labile toxin. The thin lines represent the carbon backbones. Only those (3 strands and a helices that are relevant for the active-site geometry are shown. The side chains of residues involved in the enzyme activity, especially those of the catalytic Glu and His residues, are represented by the thick lines. The a2 helix present in PT (top) and coii heat labile toxin (LT, right) is completely missing in diphtheria toxin (DT, left)...

Carroll, S. F. and Collier, R. J. (1987) Active site of Pseudomonas aeruginosa exotoxin A. Glutamic acid 553 is photolabeled by NAD and shows functional homology with glutamic acid 148 of diphtheria toxin. J. Biol. Chem. 262,8707-8711. 

Photolabeling with enzyme substrates, effectors, or photolabile analogs thereof is one of the most useful means of identifying active site residues within the primary structures of enzymes. We have recently applied this method to the study of the NAD-bind-ing sites of two mono(ADP-ribosyl) transferases, diphtheria toxin (DT) and exotoxin A (PT) from Pseudomonas aeruginosa. Both toxins (after appropriate activation steps) transfer ADP-ribose from NAD to elongation factor 2, and as a side reaction, catalyze the hydrolysis of NAD to ADP-ribose, nicotinamide, and a proton. 

Michel A, Dirkx J (1977) Occurrence of tryptophan in the enzymically active site of diphtheria toxin fragment A. Biochim Biophys Acta 491 286-295... 

Active Sites and Homology of Diphtheria Toxin and Pseudomonas aeruginosa Exotoxin A... 

Exotoxin A produced by the aerobic bacterium Pseudomonas aeruginosa (128) possesses the same catalytic activity as diphtheria toxin (120, 129), including ADP-ribosylation of EF-2 on the same site (115). The reaction is reversible because ADP-ribosylated EF-2 produced by exotoxin A can be deribosylated (functional activity restored) by incubating with excess nicotinamide and either excess exotoxin A or subunit A of diphtheria toxin. NAD+ was identified as the sole product of this reverse reaction (115). These results also establish that the configuration and site of ADP-ribosylation catalyzed by exotoxin A are identical to those of diphtheria toxin. 

Many of the differences between translation in prokaryotes and eukaryotes can be seen in the response to inhibitors of protein synthesis and to toxins. The antibiotic chloramphenicol (a trade name is Ghloromycetin) binds to the A site and inhibits peptidyl transferase activity in prokaryotes, but not in eukaryotes. This property has made chloramphenicol useful in treating bacterial infections. In eukaryotes, diphtheria toxin is a protein that interferes with protein synthesis by decreasing the activity of the eukaryotic elongation factor eEF2. 

Thus, lAP is, like diphtheria and cholera toxins, a protein toxin with an A-B structure. The holotoxin is bound to particular sites on the cell surface via its B-oligomer moiety as the first step of its interaction with mammalian cells. The A-protomer (or holotoxin itself) is then inserted to the plasma membrane traversing the lipid bilayer gradually. This slow process of the entrance of the toxin molecule is reflected in a definite lag time invariably preceding the onset of the action of lAP on intact cells as analyzed with rat pancreatic islets by kinetic and immunological approaches [25]. The A-protomer is finally activated by certain processing enzyme(s) inside the membrane to catalyze ADP-ribosylation of the target membrane protein with intracellular NAD as substrate. 

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