Prior studies have described two functionally distinct ligand-binding sites on whole diphtheria toxin, the NAD site, which catalyzes the intracellular ADP-ribosylation reaction, and the P site, which affects toxin binding to sensitive cells. Occupancy of the P site by ATP or other phosphorylated compounds inhibits toxin attachment to cells. Here we show that binding of NAD site and P site ligands is competitive; and we characterize ligand-binding properties of two mutant forms of the toxin, CRM 45 and CRM 197. The data suggest that the NAD site, on the A moiety, lies immediately adjacent to the P site, formed by a strongly cationic region on the COOH-terminal half of B. The cationic character of the P site slightly alters the substrate specificity of the NAD site, and occupancy of either of the sites blocks ligand binding to the other. Possible roles of the P site in toxin attachment are discussed.
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