1. Photoaffinity labelling of human poly(ADP-ribose) polymerase catalytic domain
- Author
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J Reinbolt, A. Ruf, J Ménissier-de Murcia, G. de Murcia, Frédéric Simonin, V. Rolli, Georg E. Schulz, H Kim, Myron K. Jacobson, University of Kentucky, Biotechnologie et signalisation cellulaire (BSC), and Université de Strasbourg (UNISTRA)-Institut de recherche de l'Ecole de biotechnologie de Strasbourg (IREBS)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Models, Molecular ,Light ,DNA Mutational Analysis ,Peptide ,MESH: Amino Acid Sequence ,Biochemistry ,MESH: Recombinant Proteins ,MESH: DNA Mutational Analysis ,MESH: Peptide Fragments ,Peptide sequence ,Polymerase ,chemistry.chemical_classification ,biology ,Chemistry ,Tryptophan ,MESH: NAD ,Affinity Labels ,Recombinant Proteins ,MESH: Mutagenesis, Site-Directed ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,Poly(ADP-ribose) Polymerases ,Sequence Analysis ,MESH: Photochemistry ,MESH: Models, Molecular ,Research Article ,Azides ,Photochemistry ,Stereochemistry ,Poly ADP ribose polymerase ,Molecular Sequence Data ,MESH: Sequence Analysis ,Labelling ,Humans ,MESH: Lysine ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Amino Acid Sequence ,Binding site ,Molecular Biology ,MESH: Tryptophan ,MESH: Affinity Labels ,MESH: Humans ,MESH: Molecular Sequence Data ,Binding Sites ,Lysine ,MESH: Poly(ADP-ribose) Polymerases ,Mutagenesis ,Cell Biology ,NAD ,MESH: Azides ,MESH: Light ,Peptide Fragments ,MESH: Binding Sites ,Mutagenesis, Site-Directed ,biology.protein ,NAD+ kinase - Abstract
Photoaffinity labelling of the human poly(ADP-ribose) polymerase (PARP) catalytic domain (40 kDa) with the NAD+ photoaffinity analogue 2-azido-[α-32P]NAD+ has been used to identify NAD+-binding residues. In the presence of UV, photoinsertion of the analogue was observed with a stoichiometry of 0.73 mol of 2-azido-[α-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 Ile879 –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, Ménissier-de Murcia, de Murcia and Schulz (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7481–7485].
- Published
- 1997