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Transition-State Structure for the ADP-Ribosylation of Recombinant G i α 1 Subunits by Pertussis Toxin

1998; American Chemical Society; Volume: 37; Issue: 9 Linguagem: Inglês

10.1021/bi972594x

1943-295X

Johannes Scheuring, Paul J. Berti, Vern L. Schramm,

Monoclonal and Polyclonal Antibodies Research

Pertussis toxin ADP-ribosylates a specific Cys side chain in the α-subunit of several G-proteins. Recombinant Giα1-subunits were rapidly ADP-ribosylated in the absence of βγ-subunits, with a Km of 800 μM and a kcat of 40 min-1. Addition of βγ-subunits decreases Km to 0.3 μM with little change of kcat. Kinetic isotope effects established the transition-state structure for ADP-ribosylation of Giα1 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 Giα1 at the transition state is greater than that for water in the hydrolysis of NAD+ by pertussis toxin. Crystal structures for Giα1 show the Cys nucleophile in a disordered segment or inaccessible for attack on NAD+. Therefore, transition-state formation requires an altered Giα1 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.