Photoaffinity labeling of terminal deoxynucleotidyl transferase. 1. Active site directed interactions with 8-azido-2'-deoxyadenosine 5'-triphosphate.
A photoaffinity analogue of dATP, 8-azido-2'-deoxyadenosine 5'-triphosphate (8-azido-dATP), was used to probe the nucleotide binding site of the non-template-directed DNA polymerase terminal deoxynucleotidyl transferase (EC 2.7.7.31). The Mg2+ form of 8-azido-dATP was shown to be an efficient enzyme substrate with a Km of 53 microM. Loss of enzyme activity occurred during UV photolysis only in the presence of 8-azido-dATP. At saturation (120 microM 8-azido-dATP), 54% of the protein molecules were modified as determined by inhibition of enzyme activity. Kinetic analysis of enzyme inhibition induced by photoincorporation of 8-azido-dATP indicated an apparent Kd of approximately 38 microM. Addition of 2 mM dATP to 120 microM 8-azido-dATP resulted in greater than 90% protection from photoinduced loss of enzyme activity. In contrast, no protection was observed with the addition of 2 mM dAMP. Enzyme inactivation was directly correlated with incorporation of radiolabeled 8-azido-dATP into the protein and UV-induced destruction of the azido group. Photoincorporation of 8-azido-dATP into terminal transferase was reduced by all purine and pyrimidine deoxynucleoside triphosphates of which dGTP was the most effective. The alpha and beta polypeptides of calf terminal transferase were specifically photolabeled by [gamma-32P]-8-azido-dATP, and both polypeptides were equally protected by all four deoxynucleoside triphosphates. This suggests that the nucleotide binding domain involves components from both polypeptides.
Evans
R K
RK
Department of Biochemistry, University of Kentucky Medical Center, Lexington 40536-0084.
Coleman
M S
MS
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism* / Q000528:radiation effects; D007700:Kinetics; D010761:Phosphorus Radioisotopes; D010782:Photolysis; D013950:Thymus Gland / Q000201:enzymology; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; D010761:Phosphorus Radioisotopes; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D004253:DNA Nucleotidylexotransferase