Biochemistry of terminal deoxynucleotidyltransferase: characterization and properties of photoaffinity labeling with 8-azidoadenosine 5'-triphosphate

Abstract

8-Azidoadenosine 5''-triphosphate (8-azido-ATP) and its photolyzed product are competitive inhibitors of terminal deoxynucleotidyltransferase [TOT, calf thymus] 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. Photoactivation of the azido-ATP-enzyme complex results in the covalent binding of azido-ATP to TDT. 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 analog was associated with the 26,000 dalton subunit of TDT. Incorporation of azido-ATP was absolutely dependent on the presence of a divalent cation. All 4 deoxyribonucleoside triphosphates as well as ATP and GTP were able to compete with azido-ATP during the incorporation experiment as judged by the competitive reduction in the cross-linking of the photoaffinity analog to 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, EDTA, .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 26,000 dalton subunit of TDT revealed that the majority of the incorporated photoaffinity analog was present in 2 peptides.