Role of tryptophan 26 in the NAD glycohydrolase reaction of the S-1 subunit of pertussis toxin.
The gene encoding a catalytically active deletion peptide, the C180 peptide, of the S-1 subunit of pertussis toxin was engineered to facilitate mutagenesis at the Trp-26 (wild-type) coding sequence. A synthetic double-stranded oligonucleotide was inserted into the C180 gene such that all possible codons would be introduced into position 26. Seven individual mutants of the C180 peptide which possessed amino acid substitutions at residue 26 (collectively termed C180W26n peptides) were purified from periplasmic extracts of Escherichia coli. Each C180W26n peptide was present as a single major peptide that had an apparent molecular mass of between 20.9 and 24.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and each showed similar immunoreactivity relative to the C180 peptide. The C180W26n peptides demonstrated marked reduction of both ADP-ribosyltransferase and NAD glycohydrolase activities at 25 nM and 10 microM NAD, respectively. Kinetic analysis of the two most active mutants, C180W26F and C180W26Y, revealed that the major perturbation of NAD glycohydrolase activity was due to an increase (approximately 20-fold) in the Km for NAD between these mutants and the C180 peptide.
Cortina
G
G
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
Barbieri
J T
JT
D001483:Base Sequence; D003062:Codon; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D037342:Pertussis Toxin; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015202:Protein Engineering; D013329:Structure-Activity Relationship; D014364:Tryptophan; D010566:Virulence Factors, Bordetella / Q000235:genetics / Q000378:metabolism*
D003062:Codon; D010566:Virulence Factors, Bordetella; D014364:Tryptophan; D011065:Poly(ADP-ribose) Polymerases; D037342:Pertussis Toxin; D009244:NAD+ Nucleosidase