The reaction mechanism for CD38. A single intermediate is responsible for cyclization, hydrolysis, and base-exchange chemistries

Human recombinant CD38 catalyzes the formation of both cyclic ADP- ribose and ADP-ribose products from NAD⁺ and hydrolyzes cyclic ADP-ribose to ADP-ribose. The corresponding GDP products are formed from NGD⁺. The enzyme was characterized by substrate and inhibition kinetics, exchange studies, rapid-quench reactions, and stopped-flow-fluorescence spectroscopy to establish the reaction mechanism and energetics for individual steps. Noncyclizable substrates NMN⁺ and nicotinamide-7-deaza-hypoxanthine dinucleotide (7-deaza NHD⁺) were rapidly hydrolyzed by the enzyme. The k(cat) for NMN⁺ was 5-fold higher than that of NAD⁺ and has the greatest reported kcat of any substrate for CD38. 7-deaza-NHD⁺ was hydrolyzed at approximately one-third the rate of NHD⁺ but does not form a cyclic product. These results establish that a cyclic intermediate is not required for substrate hydrolysis. The ratio of methanolysis to hydrolysis for cADPR and NAD+ catalyzed by CD38 increases linearly with MeOH concentration. Both reactions produce predominantly the β-methoxy riboside compound, with a relative nucleophilicity of MeOH to H₂O of 11. These results indicate the existence of a stabilized cationic intermediate for all observed chemistries in the active site of CD38. The partitioning of this intermediate between cyclization, hydrolysis, and nicotinamide-exchange unites the mechanisms of CD38 chemistries. Steady-state and pre-steady-state parameters for the partition and exchange mechanisms allowed full characterization of the reaction coordinate. Stopped-flow methods indicate a burst of cGDPR formation followed by the steady-state reaction rate. A lag phase, which was NGD⁺ concentration dependent, was also observed. The burst size indicates that the dimeric enzyme has a single catalytic site formed by two subunits. Pre- steady-state quench experiments did not detect covalent intermediates. Nicotinamide hydrolysis of NGD⁺ precedes cyclization and the chemical quench decomposes the enzyme-bound species to a mixture of cyclic and hydrolysis products. The time dependence of this ratio indicated that nicotinamide bond- breakage occurs 4 times faster than the conversion of the intermediate to products. Product release is the overall rate-limiting step for enzyme reaction with NGD⁺.