Role of histidine 35 of the S1 subunit of pertussis toxin in the ADP-ribosylation of transducin.
Molecular modeling of the S1 subunit (S1) of pertussis toxin with other ADP-ribosylating bacterial exotoxins predicted that histidine 35 (His-35) would residue within the active site of S1. Recombinant derivatives of S1 (rS1 and the C180 peptide) which contained either a H35Q or H35P mutation were analyzed to determine the role of His-35 in ADP-ribosylation. C180 peptide is a recombinant peptide composed of the amino-terminal 180 amino acids of S1. Under linear velocity conditions, C180H35Q possessed 2% of wild type C180 peptide activity and C180H35P possessed no detectable activity in the ADP-ribosylation of transducin. The H35Q mutation did not change the affinity of recombinant peptides for NAD or two targets for ADP-ribosylation, transducin, or alpha i3C20, but did lower the kcat in the NAD glycohydrolase and ADP-ribosyltransferase reactions. Neither the H35Q nor H35P mutation reduced the ability of recombinant proteins to be photocross-linked with NAD which was consistent with the His-35 mutations not reducing the affinity for NAD. These data indicate that His-35 does not reduce the affinity of S1 for NAD or transducin, but functions as a catalytic residue in the ADP-ribosylation reaction possibly in a hydrogen bonding capacity.
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Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
Barbançon-Finck
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Barbieri
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D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D006639:Histidine; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D037342:Pertussis Toxin; D017354:Point Mutation; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015293:Transducin / Q000302:isolation & purification / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D006639:Histidine; D011065:Poly(ADP-ribose) Polymerases; D037342:Pertussis Toxin; D015293:Transducin