Chemical evidence in favor of a stabilized oxocarbonium-ion intermediate in the NAD+ glycohydrolase-catalyzed reactions

Abstract The mechanism of the spontaneous solvolysis (hydrolysis and methanolysis) of the nicotinamide-ribose bond of NAD + , studied under unimolecular rate conditions, was compared to the mechanism of the reactions catalyzed by NAD + glycohydrolase. Experimental arguments—i.e., sensitivity of the observed solvolytic rates to the medium effects, stereochemical course of the methanolysis of α- and β-NAD + , partitioning ratio of the ADP-ribosyl intermediate, and entropy of activation—are presented, suggesting that the nonenzymatic solvolysis of β-NAD + proceeds through an enforced preassociation mechanism in which the acceptor molecules, methanol or (and) water, stabilize the developing ADP-ribosyl oxocarbonium ion generated by the bond cleavage. In aqueous solution this intermediate seems too unstable to exist as a fully developed solvent-equilibrated species. In contrast to the spontaneous solvolysis, the ADP-ribosyl intermediate generated in the active site of NAD + glycohydrolase shows a high selectivity when reacting with competing acceptor nucleophiles. This suggests that the enzyme provides a substantial stabilization of the intermediary ADP-ribosyl oxocarbonium ion. We conclude that catalysis of the nicotinamide-ribose bond cleavage by NAD + glycohydrolase would be due, in part, to the ability of the enzyme to effectively stabilize an oxocarbonium ion-like structure in the transition state.