Transition-state structure for the ADP-ribosylation of recombinant Gialpha1 subunits by pertussis toxin.
Pertussis toxin ADP-ribosylates a specific Cys side chain in the alpha-subunit of several G-proteins. Recombinant Gialpha1-subunits were rapidly ADP-ribosylated in the absence of betagamma-subunits, with a Km of 800 microM and a kcat of 40 min-1. Addition of betagamma-subunits decreases Km to 0.3 microM with little change of kcat. Kinetic isotope effects established the transition-state structure for ADP-ribosylation of Gialpha1 subunits. The transition state is dissociative, with a 2.1 A bond to the nicotinamide leaving group and a bond of 2.5 A to the sulfur nucleophile. The nucleophilic participation of Gialpha1 at the transition state is greater than that for water in the hydrolysis of NAD+by pertussis toxin. Crystal structures for Gialpha1 show the Cys nucleophile in a disordered segment or inaccessible for attack on NAD+. Therefore, transition-state formation requires an altered Gialpha1 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.
Scheuring
J
J
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Berti
P J
PJ
Schramm
V L
VL
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D002384:Catalysis; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D010566:Virulence Factors, Bordetella; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go