Adenosine Diphosphate Ribosylation of Aminoacyl Transferase II and Inhibition of Protein Synthesis by Diphtheria Toxin

Abstract

Abstract Diphtheria toxin catalyzes the transfer of the adenosine diphosphate ribose portion of nicotinamide adenine dinucleotide to aminoacyl transferase II purified to a homogeneous state from rat liver. A stoichiometric amount of nicotinamide is released simultaneously. The ADP-ribosylation of aminoacyl transferase II results in a concomitant inactivation of this particular enzyme, which has a function in the translocation of peptidyl transfer RNA on ribosomes. The reaction is reversible, since toxin catalyzes the formation of NAD from ADP-ribosylated aminoacyl transferase II and nicotinamide with a concurrent restoration of the enzyme activity. The equilibrium constant of the ADP-ribosylation reaction is determined to be 6.3 x 10-4. From this constant the free energy of hydrolysis of the ADP-ribose transferase II linkage is calculated to be approximately 4000 cal per mole at pH 7 and 25°. The ADP-ribosylation and reverse reactions are most active at pH 8.5 and 5.2, respectively. The apparent Michaelis constants for NAD and nicotinamide are 5 x 10-6 m at pH 7.4 and 5 x 10-4 m at pH 5.2, respectively. No other derivatives of NAD so far tested are capable of replacing NAD except thionicotinamide adenine dinucleotide. Diphtheria antitoxin inhibits the reaction, and toxoid is inactive. So far aminoacyl transferase II is the sole protein species found to be ADP-ribosylated in the supernatant fraction from rat liver. Toxin catalyzes neither hydrolysis of NAD nor the exchange reaction between NAD and nicotinamide-14C.