Dissection of ADP-ribose polymer synthesis into individual steps of initiation, elongation, and branching.
We have studied the automodification reaction of poly(ADP-ribose)polymerase (PARP) (EC 2.4.2.30). The individual reactions of initiation, elongation, and branching catalyzed by this enzyme have been dissected out by manipulating the concentration of beta NAD, the ADP-ribosylation substrate. While PARP-mono(ADP-ribose) conjugates were the predominant products of automodification at 200 nM NAD (initiation), highly branched and complex polymers were synthesized at 200 microM NAD (polymerization). Initial rates of automodification increased with second order kinetics as a function of the enzyme concentration at both 200 nM and 200 microM NAD. These results are consistent with the conclusion that two molecules of PARP are required for ADP-ribose polymer synthesis during enzyme automodification. Thus, the auto-poly(ADP-ribosyl)ation reaction of PARP is intermolecular. In agreement with this notion, we observed that initial rates of the initiation reaction with 3'-deoxyNAD as a substrate also increased with the square of the enzyme concentration. In addition, the auto-poly(ADP-ribosyl)ation reaction of PARP increased with second order kinetics as a function of the NAD concentration at nanomolar levels (0.2-106 microM). Therefore, the dimeric structure of PARP also requires two molecules of bound NAD for efficient ADP-ribose polymerization.
Alvarez-Gonzalez
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R
Department of Microbiology and Immunology, University of North Texas Health Science Center at Fort Worth 76107-2699, USA.
Mendoza-Alvarez
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H
D002384:Catalysis; D007700:Kinetics; D015394:Molecular Structure; D010441:Peptide Chain Elongation, Translational; D010442:Peptide Chain Initiation, Translational; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis* / Q000737:chemistry; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*
D011064:Poly Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases