DeoxyNAD and deoxyADP-ribosylation of proteins

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Abstract
Recently, two deoxyribose analogs of βNAD+ (2′-deoxy and 3′-deoxyNAD+) have been synthesized and purified in this laboratory. Whereas 2′-deoxyNAD+ was an efficient substrate for arg-specific mon(ADP-ribosyl) transferases, it was not a substrate for poly(ADP-ribose) polymerase (PARP). Instead, it was a non-competitive inhibitor of βNAD+ in the ADP-ribose polymerization reaction catalyzed by PARP. Thus, 2′-deoxyNAD+ has been utilized to distinguish between mono(ADP-ribose) and poly(ADP-ribose) acceptor proteins. 2′-deoxyNAD+ has also been used to characterize the arg-specific mono(2′-deoxyADP-ribosyl)ation reaction of PARP with cholera toxin or avian mono(ADP-ribosyl)transferase. By contrast, 3′-deoxyNAD+ can effectively be utilized as a substrate by PARP. However, while the estimated Km and Kcat of polymerization with 3′-deoxyNAD+ can were 20 μM and 0.11 moles/sec, the Km and Kcat with βNAD+ as a substrate were 59 μM and 1.29 moles/sec, respectively. Determination of the average size of 3′-deoxyADP-ribose polymers indicated that chains no larger than four residues are synthesized with this substrate. Thus, the utilization of 3′-deoxyNAD+ has facilitated the electrophoretic identification of poly(ADP-ribose) acceptor proteins in mammalian chromatin.