Kinetic isotope effect studies on the formation and hydrolysis of cADPR by CD38A

The lymphocyte protein ('I)38 has been shown lo Gill alvze tile trormation of cyclic ADP ribose (cADPR) from NAD+ and lo hydrolvze cADPtt and NAD+ to ADP-ribose (.DPR). The goals of this projecl are to characterize the three transition state structures for the three chemically distincl reactions catalyzed by CD38. Kinetic isotope el[acts tRIEs tirh, drolysis of radiolabeh'd sub strates cADPR and NAD+ were determined at 37, of and pll 7.5 in 50 mM phosphate buffer for soluble recombinant (7I)38. Kldestaer hevdrolysis of NAD+ labeled with l'-:ttl and l' HC were 1.010 and too respectively, demonstrating that chemistry is not rate limiting for the reaction, l,abeled cADPR was synthesized with ADP-ribosylcyclase and isotopically labeled NAD+. Kinetic isotope effects obtained for cADPR hydrolysis indicated rate-limiting chemistry by the relalively large KIEs of tilt (1.26). 1-24 (1.050). and 5'-61 (l.051). The modest iLI4 KIE arid the large are consistent with an oxocarbenium-ion transition state. Studies el methanolysis of NAD+ and cADIR suggest that a stabilized cation at the active, lite precedes ADPI/for marion. A relative nucleophilicily of 10/l: MeOII/H20 in the product ratio for methoxy-ADPR/A1)PR for both reactions suggests an intermediate lifetime which dermits solvent equilibration. Only' .#methoxy ADPII. was detected as a product of methanolysis indicating that hydrolysis likely proceeds with retention of configuration at (21'. The methanolysis results implicate a common intermediate toward ADPIC formation starting from till her.