Covalent binding of 4-carbamoylbenzenediazonium chloride to deoxyguanine bases of DNA resulting in apparent irreversible inhibition of poly(adenosine diphosphoribose) polymerase at the nicotinamide binding site

Abstract

The poly(adenosine diphosphoribose) polymerase activity of isolated liver nuclei was inhibited by 4-carbamoylbenzenediazonium chloride, referred to as 4-diazoniobenzamide, an effect that was dependent on the time of incubation and the concentration of the diazonium compound, with inhibition following first-order kinetics. The inhibition was not reversed by reisolation of nuclei and centrifugal washing, whereas the inhibition by benzamide or 4-aminobenzamide was completely reversible under these conditions. Simultaneous incubation of 4-diazoniobenzamide with benzamide prevented enzyme inhibition. The 4-diazoniobenzoic acid analogue was not inhibitory. The mechanism of action of 4-diazoniobenzamide was traced to a specific covalent binding to dGMP of DNA to form N2-[(4-carbamoylphenyl)azo]-2'-deoxyguanosine 5'-monophosphate. Coenzymic DNA, by tight association with the polymerase protein, fixes the -C(O)NH2 moiety of the adduct at the nicotinamide-binding site of the enzyme.

Diazo Group Electrophilicity in Kinamycins and Lomaiviticin A: Potential Insights into the Molecular Mechanism of Antibacterial and Antitumor Activity

“…In the reactions of aryldiazonium ions with DNA, radical formation is preceded by nucleophilic attack by the C-2 amino group of guanine residues and by the C-6 amino group of adenine to form labile triazenes. Such intermediates, which are isolable in some cases, decompose with loss of N 2 to form aryl radicals which combine with the purine ring radical to form C-8 aryl purines and also react in other ways with the initially modified DNA molecule to form depurinated sites as well as to cleave the phosphodiester backbone.

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Molecular Cloning of Adenosinediphosphoribosyl Transferase.

“…shows competition with respect to NAD (K I 2 0.5 pM). Various forms of mixed type inhibitions can be observed at widely varying concentrations of the dye, as would be anticipated also from the + close vicinity of the NAD and DNA binding sites on the enzyme, which was calculated to be 8.5 A (24), and from apparently allosteric mechanisms (4). The mechanism of the obligatory coenzymic function of DNA, that binds tightly to the transferase (7,19,23),…”