Mechanism of cyclization of pyridine nucleotides by bovine spleen NAD+ glycohydrolase.

We have shown that bovine spleen NAD+ glycohydrolase (EC), purified to homogeneity, is a multifunctional enzyme. A time-dependent formation of cADPR from NAD+ that did not exceed 1.5-2% of the reaction products was measurable. The cyclase activity of this enzyme was, however, best evidenced by its transformation of NGD+ into cyclic GDP-ribose (cGDPR). The formation of the cyclic compound could be monitored spectroscopically (UV and fluorescence) and by high-performance liquid chromatography; the product ratio of cGDPR/GDP-ribose was 2:1. Bovine spleen NAD+ glycohydrolase is also able to hydrolyze cADPR (Muller-Steffner et al. (1994) Biochem. Biophys. Res. Commun. 204, 1279-1285); the kinetic parameters (V/Km) measured exclude, however, the possibility that cADPR is a kinetically competent reaction intermediate in the transformation of NAD+ into ADP-ribose. Experimental data indicating that NAD+ glycohydrolase-catalyzed hydrolysis and methanolysis of NA(G)D+ occurred at the expense of the formation of the cyclic compounds are in favor of a reaction mechanism involving the partitioning of a common oxocarbenium reaction intermediate between the different acceptors. Thus E.A(G)DP-ribosyl oxocarbenium intermediate can react according to i) intramolecular processes with the positions N-1 of adenine and N-7 of guanine to give cA(G)DPR as reaction products, and ii) intermolecular reactions with water (formation of A(G)DP-ribose) and methanol (formation of methyl A(G)DP-ribose). We attribute the marked difference in yield of cADPR and cGDPR to the intrinsic reactivity (nucleophilicity and positioning) of the purine N-positions that are involved in the cyclization reactions within the E.A(G)DP-ribosyl oxocarbenium complexes.