Biochemistry of terminal deoxynucleotidyltransferase: identification, characterization, requirements, and active-site involvement in the catalysis of associated pyrophosphate exchange and pyrophosphorolytic activity.
Terminal deoxynucleotidyltransferase (TdT) has been found to catalyze both pyrophosphate exchange and pyrophosphorolysis reactions. Both reactions are strongly inhibited by antiserum to TdT. The reactions require the presence of a divalent cation, a single- or double-stranded oligomeric or polymeric DNA or RNA, and deoxyribonucleoside triphosphates (for PPi exchange only). Of the three divalent cations tested, Mg2+ and Co2+ are equally effective, while Mn2+ neither is used for catalysis nor inhibits the Mg2+-catalyzed reactions. Ribonucleoside triphosphates have been found to support the PPi exchange reaction to a minor extent and have no inhibitory effect on the catalysis mediated by dNTPs. Inhibition studies, using SH group inhibitors, Zn chelator, and a substrate binding site specific reagent, revealed that PPi exchange and pyrophosphorolysis reactions may be distinguished by differences in their sensitivity to inhibition by various reagents. While the PPi exchange reaction is strongly inhibited by sulfhydryl reagents, o-phenanthroline, and pyridoxal phosphate, the pyrophosphorolysis reaction is insensitive to these reagents. In addition, the pyrophosphorolysis reaction is also found not to require a free 3'-OH terminus of a primer. This difference in the susceptibility of the two reactions indicates that discrete active-site structures exist in TdT which catalyze PPi exchange and pyrophosphorolysis reactions.
D001665:Binding Sites; D002413:Cations, Divalent; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D011756:Diphosphates; D007106:Immune Sera; D007118:Immunoassay; D007700:Kinetics; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002413:Cations, Divalent; D011756:Diphosphates; D007106:Immune Sera; D013439:Sulfhydryl Reagents; D004254:DNA Nucleotidyltransferases