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.
Muller-Steffner
H M
HM
Laboratoire de Chimie Bioorganique, Laboratoire Associé au Centre National de la Recherche Scientifique 1386, Faculté de Pharmacie, Université Louis Pasteur Strasbourg, 74 route du Rhin, 67400 Illkirch, France.
Augustin
A
A
Schuber
F
F
D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D036563:Cyclic ADP-Ribose; D006156:Guanosine Diphosphate Sugars / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology; D013379:Substrate Specificity
D006156:Guanosine Diphosphate Sugars; C090657:cyclic guanosine diphosphate-ribose; D009243:NAD; D036563:Cyclic ADP-Ribose; D000246:Adenosine Diphosphate Ribose; D009244:NAD+ Nucleosidase