Photoaffinity labelling of human poly(ADP-ribose) polymerase catalytic domain.
Photoaffinity labelling of the human poly(ADP-ribose) polymerase (PARP) catalytic domain (40 kDa) with the NAD+ photoaffinity analogue 2-azido-[alpha-32P]NAD+ has been used to identify NAD+-binding residues. In the presence of UV, photo-insertion of the analogue was observed with a stoichiometry of 0.73 mol of 2-azido-[alpha-32P]NAD+ per mol of catalytic domain. Competition experiments indicated that 3-aminobenzamide strongly protected the insertion site. Residues binding the adenine ring of NAD+ were identified by trypsin digestion and boronate affinity chromatography in combination with reverse-phase HPLC. Two major NAD+-binding residues, Trp1014 of peptide Thr1011-Trp1014 and Lys893 of peptide Ile979-Lys893, were identified. The site-directed mutagenesis of these two residues revealed that Lys893, but not Trp1014, is critical for activity. The close positioning of Lys893 near the adenine ring of NAD+ has been confirmed by the recently solved crystallographic structure of the chicken PARP catalytic domain [Ruf, Menissier-de Murcia, de Murcia and Schulz (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7481-7485].
Kim
H
H
Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington 40536-0082, U.S.A.
Jacobson
M K
MK
Rolli
V
V
Ménissier-de Murcia
J
J
Reinbolt
J
J
Simonin
F
F
Ruf
A
A
Schulz
G
G
de Murcia
G
G
D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D004252:DNA Mutational Analysis; D006801:Humans; D008027:Light; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010777:Photochemistry; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009243:NAD; C062879:nicotinamide 2-azidoadenine dinucleotide; D014364:Tryptophan; D011065:Poly(ADP-ribose) Polymerases; D008239:Lysine