A proposed mechanism of ADP-ribosylation catalyzed by the pertussis toxin S1 subunit.
Pertussis toxin is a complex protein composed of five different subunits, named S1 through S5 and arranged in an A-B structure. The B oligomer, composed of S2 through S5, is the receptor-binding moiety, and the A promoter, composed of S1, is the enzymatically active moiety. S1 catalyzes the ADP-ribosylation of a cysteine in the alpha subunit of heterotrimeric G proteins. In the absence of G proteins it also catalyzes the cleavage of NAD+ into ADP-ribose and nicotinamide. Molecular dissection has indicated that the C-terminal domain of S1 is involved in G-protein binding, while the N-terminal domain, homologous to other ADP-ribosylating toxins, contains the NAD(+)-binding site and the residues involved in catalysis. By site-directed mutagenesis and kinetic analyses Glu-129 and His-35 were identified as the catalytic residues. Glutamates analogous to Glu-129 are found in all studied ADP-ribosylating toxins, while His-35 is less well conserved. This suggests that Glu-129 acts on the common substrate NAD+, whereas His-35 plays its role on the acceptor substrates. We propose a mechanism in which Glu-129 exerts its action on the 2'-OH group of the NAD+ ribose, thereby facilitating the formation of an oxocarbonium-like intermediate and the weakening of the N-glycosidic bond. His-35 could increase the nucleophilicity of the cysteine in the G protein or the water molecule to attack the weakened N-glycosidic bond of NAD+ and yield the products of the reaction.
Locht
C
C
Laboratoire de Microbiologie Génétique et Moléculaire, INSERM CJF-9109, Institut Pasteur de Lille, France.
Antoine
R
R
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism; D001665:Binding Sites; D001886:Bordetella pertussis / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D037342:Pertussis Toxin; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D010566:Virulence Factors, Bordetella / Q000737:chemistry* / Q000378:metabolism*
D001427:Bacterial Toxins; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; C083388:pertussis toxin, S1 subunit; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins