Biochemistry of terminal deoxynucleotidyltransferase (TdT): characterization and mechanism of inhibition of TdT by P1, P5-bis(5'-adenosyl) pentaphosphate.
The catalysis of DNA synthesis by calf thymus terminal deoxynucleotidyltransferase (TdT) is strongly inhibited in the presence of Ap5A, while replicative DNA polymerases from mammalian, bacterial, and oncornaviral sources are totally insensitive to Ap5A addition. The Ap5A-mediated inhibition of TdT seems to occur via its interaction at both the substrate binding and primer binding domains as judged by classical competitive inhibition plots with respect to both substrate deoxynucleoside triphosphate (dNTP) and DNA primer and inhibition of ultraviolet light mediated cross-linking of substrate dNTP and oligomeric DNA primer to their respective binding sites. Further kinetic analyses of Ap5A inhibition revealed that the dissociation constant of the Ap5A-enzyme complex, with either substrate binding or primer binding domain participating in the complex formation, is approximately 6 times higher (Ki = 1.5 microM) compared to the dissociation constant (Ki = 0.25 microM) of the Ap5A-TdT complex when both domains are available for binding. In order to study the binding stoichiometry of Ap5A to TdT, an oxidized derivative of Ap5A, which exhibited identical inhibitory properties as its parent compound, was employed. The oxidation product of Ap5A, presumably a tetraaldehyde derivative, binds irreversibly to TdT when the inhibitor-enzyme complex is subjected to borohydride reduction. The presence of aldehyde groups in the oxidized Ap5A appeared essential for inhibitory activity since its reduction to alcohol via borohydride reduction or its linkage to free amino acids prior to use as an inhibitor rendered it completely ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)
D000227:Adenine Nucleotides / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D004253:DNA Nucleotidylexotransferase / Q000037:antagonists & inhibitors*; D004254:DNA Nucleotidyltransferases / Q000037:antagonists & inhibitors*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D015226:Dinucleoside Phosphates; D007700:Kinetics; D011485:Protein Binding; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D013950:Thymus Gland / Q000201:enzymology
D000227:Adenine Nucleotides; D015226:Dinucleoside Phosphates; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; D004254:DNA Nucleotidyltransferases; D004253:DNA Nucleotidylexotransferase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase