ADP-ribosylation by Clostridium botulinum C3 exoenzyme increases steady-state GTPase activities of recombinant rhoA and rhoB proteins.

ADP-ribosylation of recombinant rhoA and rhoB proteins by Clostridium botulinum C3 exoenzyme increased steady-state GTP hydrolysis by 50 to 80%. ADP-ribosylation and increase in GTP hydrolysis occurred at similar concentrations of C3, depended on the presence of NAD and were prevented by anti-C3 antibody or heat inactivation of C3. In contrast, GTP hydrolysis by Ile-41 rhoA or Ha-ras, which are no substrates for the transferase, were not affected by C3. ADP-ribosylation facilitated the [3H]GDP release and subsequently, the binding of [3H]GTP to rhoA. The data indicate that the increase in the steady-state GTPase activity by ADP-ribosylation is caused by increasing the rate of GDP release which is suggested to be the rate limiting step of the GTPase cycle of the small GTP-binding proteins.