Covalent Alterations of Protein Structure in the Inhibition of Protein Synthesis by Diphtheria Toxin

Abstract

The toxicity of diphtheria toxin is believed to result from its activity in inhibiting protein synthesis in sensitive cells. This activity is now known to involve two distinct examples of covalent modification of protein structure. a) The toxin (m.wt. 63,000) inhibits protein synthesis by catalyzing a covalent modification of elongation factor 2 (EF2). The modification involves attachment of the adenosine diphosphate ribose (ADPR) portion of NAD+ to the factor, with concomitant release of nicotinamide and a proton. The modified factor can no longer function in the translocation process on ribosomes, perhaps because of its altered capacity to interact with RNA. b) The intact toxin molecules, a single 63,000 dalton chain, is inactive in catalyzing the ADP-ribosylation of EF 2, and must be subjected to limited proteolysis and reduction for this activity to be expressed. Trypsin, which is the most effective protease tested, attacks the toxin at any of at least three closely spaced arginines, splitting it into two large fragments, A (m.wt. 24,000) and B (m.wt. 39,000) linked by a disulfide bridge. Free fragment A, released after reduction of this disulfide, is responsible for the enzymic activity of the toxin.