Poly(ADP-ribose) polymerase is a catalytic dimer and the automodification reaction is intermolecular.

Abstract

We have determined the molecular mechanism of the automodification reaction of poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30). While PARP-mono(ADP-ribose) conjugates were the predominant products of automodification at 200 nM NAD, enzyme-bound branched polymers were preferentially synthesized at 200 microM NAD. Thus, the initiation, elongation, and branching reactions catalyzed by PARP appear to be [NAD]-dependent. Initial rates of automodification increased with second order kinetics as a function of [PARP] at both 200 nM and 200 microM NAD. Therefore, 2 molecules of PARP, i.e. a catalytic dimer, are required for the auto-mono(ADP-ribosyl)ation and the auto-poly(ADP-ribosyl)ation reactions. Initial rates of automodification also increased with second order kinetics at low NAD concentrations. Therefore, the catalytic dimer also requires 2 molecules of NAD. These results are consistent with the conclusion that the automodification reaction of PARP is intermolecular and that the 2 monomeric units of PARP may simultaneously function as catalyst and acceptor molecules in the automodification reaction. Confirmatory evidence for the catalytic role of protein-protein interactions in the automodification reaction was manifested by a marked inhibition of auto-poly(ADP-ribosyl)ation at 40 nM or higher [PARP].