Biochemistry of terminal deoxynucleotidyltransferase (TdT): characterization and mechanism of inhibition of TdT by P1,P5-bis(5'-adenosyl) pentaphosphate

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Abstract
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 (a) classical competitive inhibition plots with respect to both substrate deoxynucleoside triphosphate (dNTP) and DNA primer and (b) 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 .mu.M) compared to the dissociation constant (Ki = 0.25 .mu.M) 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. With use of a tritiated oxidation product of Ap5A, a binding stoichiometry of 1 mol of Ap5A to 1 mol of TdT was observed. Thus, a single Ap5A molecule seems to substrate and primer binding site domains in TdT.