Photoaffinity labeling of terminal deoxynucleotidyl transferase. 2. Identification of peptides in the nucleotide binding domain.
Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the nucleotide binding site by the substrate photoaffinity analogue [gamma-32P]-8-azido-dATP. The alpha and beta polypeptides of photolabeled terminal transferase were resolved by high-performance liquid chromatography. The beta polypeptide was digested with trypsin and fractionated by reverse-phase chromatography. Two 32P-containing fractions were isolated and subjected to amino acid sequence analysis. Peptides were identified as Ile209-Lys232 (B26) and Val233-Lys239 (B27). Peptide B26 was further resolved into two overlapping species; one contained an additional lysine residue at the N-terminus which resulted from tryptic cleavage between Lys207 and Lys208. In order to ensure that the sequenced peptides corresponded to the photolabeled species, we devised an anion-exchange procedure to isolate photolabeled peptides from the mixture. Analysis of photolabeled peptides from terminal transferase alpha beta using DEAE-cellulose chromatography followed by reverse-phase HPLC confirmed that the photolabeled species were peptides B26 and B27. Peptide B26, the major photolabeled species, contained a conserved octapeptide region found in several eucaryotic DNA polymerases. In addition, peptide B27 was flanked by a sequence that has been implicated in triphosphate binding in other proteins. Structure predictions, based on sequence data, place the two peptides identified by photolabeling in spatial proximity consistent with the participation of both in the nucleotide binding domain.
Evans
R K
RK
Department of Biochemistry, University of Kentucky, Lexington 40536-0084.
Beach
C M
CM
Coleman
M S
MS
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D013950:Thymus Gland / Q000201:enzymology; D014357:Trypsin
D000345:Affinity Labels; D001386:Azides; D010446:Peptide Fragments; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D004253:DNA Nucleotidylexotransferase; D014357:Trypsin