Biochemistry of terminal deoxynucleotidyltransferase: characterization and properties of photoaffinity labeling with 8-azidoadenosine 5'-triphosphate.
We have found that 8-azidoadenosine 5'-triphosphate (8-azido-ATP) and its photolyzed product are competitive inhibitors of terminal deoxynucleotidyltransferase with respect to substrate deoxynucleoside triphosphates. A detailed characterization of the inhibitory effect of 8-azido-ATP revealed that its mechanism of inhibition is identical with that reported for ATP [Modak, M. J. (1978) Biochemistry 17, 3116-3120]. Photoactivation of the azido-ATP-enzyme complex results in the covalent binding of azido-ATP to terminal deoxynucleotidyltransferase. No significant incorporation of prephotolyzed azido-ATP or unsubstituted ATP into enzyme protein is noted when complexes of these nucleotides with enzyme were exposed to identical photoactivation conditions. The majority of incorporated analogue was associated with the 26 000-dalton subunit of terminal deoxynucleotidyltransferase. Incorporation of azido-ATP was further found to be absolutely dependent on the presence of a divalent cation. All four deoxyribonucleoside triphosphates as well as ATP and guanosine 5'-triphosphate were able to compete with azido-ATP during the incorporation experiment as judged by the competitive reduction in the cross-linking of the photoaffinity analogue to terminal deoxynucleotidyltransferase (TDT). In addition, substrate binding site directed inhibitors, pyrophosphate and pyridoxal 5'-phosphate, effectively blocked the incorporation of azido-ATP into enzyme protein, while several other inhibitors of TDT catalysis, namely, ethylenediaminetetraacetic acid, alpha, alpha'-dipyridyl, 1,10-phenanthroline, p-(chloromercuri)benzoate, Rose Bengal, and the presence of 0.5 M KCl, influenced the cross-linking reaction to varying degrees. A tryptic peptide analysis of the azido-ATP-labeled 26K subunit of TDT revealed that the majority of the incorporated photoaffinity analogue was present in two peptides.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D002417:Cattle; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D007700:Kinetics; D010761:Phosphorus Radioisotopes; D010782:Photolysis; D013950:Thymus Gland / Q000201:enzymology
D000345:Affinity Labels; D001386:Azides; D003848:Deoxyguanine Nucleotides; D010761:Phosphorus Radioisotopes; C017040:8-azidoadenosine 5'-triphosphate; C029603:deoxyguanosine triphosphate; D000255:Adenosine Triphosphate; D004254:DNA Nucleotidyltransferases; D004253:DNA Nucleotidylexotransferase