Studies on diphtheria toxin the effect of GTP on the toxin-dependent adenosine diphosphate ribosylation of rat liver aminoacyl transferase II

Abstract

1. (1)Diphtheria toxin catalyzes the transfer of ADP-ribose from NAD to aminoacy transferase II (T2). The resulting ADP-ribosyl-T2 complex is devoid of enzymatic activity: both the translocase and the ribosome-dependent GTPase activities are inhibited. GTP protects T2 from ADP-ribosylation. 2. (2)By equilibrium dialysis the dissociation constants and the molarity of the T2 protein groups interacting with ADP-ribose and with GTP have been established. The same number of binding sites is present for the two ligands. The K d of the GTP-T2 complex is 4.2·10 −7 M and that of the ADP-ribosyl-T2 complex is 4.9·10 −7 M. 3. (3)While GTP lowers the affinity of T2 for ADP-ribose, the presence of diphtheria toxin and NAD does not affect the binding of GTP to T2; thus the binding sites for GTP and for ADP-ribose are not the same. 4. (4)GTP binds to diphtheria toxin with a K d = 5.9·10 −6 M and behaves as a competitive inhibitor of the toxin-NAD interaction. Considering that GTP binds both to diphtheria toxin and to transferase, the mechanism by which the guanosine nucleotide protects T2 from ADP-ribosylation is discussed.