Transition-state structure for the ADP-ribosylation of recombinant G(iα1) subunits by pertussis toxin

Pertussis toxin ADP-ribosylates a specific Cys side chain in the α- subunit of several G-proteins. Recombinant G(iα1)-subunits were rapidly ADP- ribosylated in the absence of βγ-subunits, with a K(m) of 800 μM and a k(cat) of 40 min^-1. Addition of βγ-subunits decreases K(m) to 0.3 μM with little change of k(cat). Kinetic isotope effects established the transition-state structure for ADP-ribosylation of G(iα1) subunits. The transition state is dissociative, with a 2.1 Å bond to the nicotinamide leaving group and a bond of 2.5 Å to the sulfur nucleophile. The nucleophilic participation of G(iα1) at the transition state is greater than that for water in the hydrolysis of NAD⁺ by pertussis toxin. Crystal structures for G(iα1) show the Cys nucleophile in a disordered segment or inaccessible for attack on NAD⁺. Therefore, transition-state formation requires an altered G(iα1) conformation to expose and ionize Cys. The transition state has been docked into the crystal structure of pertussis toxin in a geometry required for transition state formation.