Catalytic activities of synthetic octadeoxyribonucleotides as coenzymes of poly(ADP-ribose) polymerase and the identification of a new enzyme inhibitory site

Abstract

The catalytic activity of highly purified poly(ADP-ribose) polymerase was determined at constant NAD + concentration and varying concentrations of sDNA or synthetic octadeoxyribonucleotides of differing composition. The coenzymic activities of deoxyribonucleotides were compared in two ways: (i) graphic presentation of the activation of poly(ADP-ribose) polymerase in the presence of a large concentration range of deoxyribonucleotides and (ii) by calculating k D values for the deoxyribonucleotides. As determined by method i, auto-mono-ADP-ribosylation of the enzyme protein at 25 nM NAD + was maximally activated at 1:1 octamer/enzyme molar ratios by the octadeoxyribonucleotide derived from the regulatory region of SV40 DNA (duplex C). At a 0.4:1 sDNA/enzyme ratio, sDNA was the most active coenzyme for monoADP-ribosylation. At 200 μM NAD +, resulting in polymer synthesis and with histones as secondary polymer acceptors, duplex C was the most active coenzyme, and the octamer containing the steroid hormone receptor binding consensus sequence of DNA was a close second, whereas sDNA exhibited an anomalous biphasic kinetics. sDNA was effective on mono- ADP-ribosylation at a concentration 150–200-times lower than on polymer formation. When comparison of deoxyribonucleotides was based on method ii ( k D values), by far the most efficiently binding coenzyme for both mono and polymer synthesis was sDNA, followed by duplex C, with (dA-dT) 8 exhibiting the weakest binding. The synthetic molecule 6-amino-1,2-benzopyrone (6-aminocoumarin) competitively inhibited the coenzymic function of synthetic octadeoxyribonucleotides at constant concentration of NAD +, identifying a new inhibitory site of poly(ADP-ribose) polymerase.