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 othe r ADP-ribosylating bacterial exotoxins predicted that histidine 35 (Hi s-35) would reside within the active site of S1. Recombinant derivativ es of S1 (rS1 and the C180 peptide) which contained either a H35Q or H 35P mutation were analyzed to determine the role of His-35 in ADP-ribo sylation. C180 peptide is a recombinant peptide composed of the amino- terminal 180 amino acids of S1. Under linear velocity conditions, C180 H35Q possessed 2% of wild type C 180 peptide activity and C180H35P pos sessed no detectable activity in the ADP-ribosylation of transducin. T he H35Q mutation did not change the affinity of recombinant peptides f or NAD or two targets for ADP-ribosylation, transducin, or alpha(i3)C2 0, but did lower the k(cat) in the NAD glycohydrolase and ADP-ribosylt ransferase reactions. Neither the H35Q nor H35P mutation reduced the a bility 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.