Identification of an active-site residue in subunit S1 of pertussis toxin by photocrosslinking to NAD.
The site of interaction of NAD with the isolated S1 subunit of pertussis toxin was investigated by photoaffinity labelling. When S1 was irradiated at 254 nm in the presence of [carbonyl-14C]- or [adenine-14C]NAD, the uptake of radioactivity was equivalent to 0.75 and 0.1 mol/mol respectively, while the NAD glycohydrolase activity was abolished. Inactivation was thus accompanied by crosslinking of the nicotinamide portion of NAD to the protein. Sequence determination of purified radioactive peptides indicated that Glu-129 was a major site of labelling. This residue is therefore closely associated with either NAD binding or hydrolysis.
Cockle
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Connaught Centre for Biotechnology Research, Willowdale, Ontario, Canada.
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D037342:Pertussis Toxin; D010777:Photochemistry; D013056:Spectrophotometry, Ultraviolet; D010566:Virulence Factors, Bordetella / Q000235:genetics / Q000378:metabolism*
D003432:Cross-Linking Reagents; D010566:Virulence Factors, Bordetella; D009243:NAD; D037342:Pertussis Toxin