Photolabelling of mutant forms of the S1 subunit of pertussis toxin with NAD+.
The S1 subunit of pertussis toxin catalyses the hydrolysis of NAD+ (NAD+ glycohydrolysis) and the NAD(+)-dependent ADP-ribosylation of guanine-nucleotide-binding proteins. Recently, the S1 subunit of pertussis toxin was shown to be photolabelled by using radiolabelled NAD+ and u.v.; the primary labelled residue was Glu-129, thereby implicating this residue in the binding of NAD+. Studies from various laboratories have shown that the N-terminal portion of the S1 subunit, which shows sequence similarity to cholera toxin and Escherichia coli heat-labile toxin, is important to the maintenance of both glycohydrolase and transferase activity. In the present study the photolabelling technique was applied to the analysis of a series of recombinant-derived S1 molecules that possessed deletions or substitutions near the N-terminus of the S1 molecule. The results revealed a positive correlation between the extent of photolabelling with NAD+ and the magnitude of specific NAD+ glycohydrolase activity exhibited by the mutants. Enzyme kinetic analyses of the N-terminal mutants also identified a mutant with substantially reduced activity, a depressed photolabelling efficiency and a markedly increased Km for NAD+. The results support a direct role for the N-terminal region of the S1 subunit in the binding of NAD+, thereby providing a rationale for the effect of mutations in this region on enzymic activity.
Cieplak
W
W
Laboratory of Pathobiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT 59840.
Locht
C
C
Mar
V L
VL
Burnette
W N
WN
Keith
J M
JM
D046911:Macromolecular Substances; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D009244:NAD+ Nucleosidase; D037342:Pertussis Toxin; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays; D010566:Virulence Factors, Bordetella / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D009243:NAD; D037342:Pertussis Toxin; D009699:N-Glycosyl Hydrolases; D009244:NAD+ Nucleosidase