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-ribo se and ADP-ribose products from NAD(+) and hydrolyzes cyclic ADP-ribos e to ADP-ribose. The corresponding GDP products are formed from NGD(+) . The enzyme was characterized by substrate and inhibition kinetics, e xchange studies, rapid-quench reactions, and stopped-flow-fluorescence spectroscopy to establish the reaction mechanism and energetics for i ndividual steps. Noncyclizable substrates NMN+ and nicotinamide-7-deaz a-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 k(cat) of any substrate for CD38, 7-deaz a-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 me thanolysis to hydrolysis for cADPR and NAD(+) catalyzed by CD38 increa ses linearly with MeOH concentration. Both reactions produce predomina ntly the beta-methoxy riboside compound, with a relative nucleophilici ty of MeOH to H2O of 11. These results indicate the existence of a sta bilized cationic intermediate for all observed chemistries in the acti ve site of CD38. The partitioning of this intermediate between cycliza tion, hydrolysis, and nicotinamide-exchange unites the mechanisms of C D38 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 f ormation followed by the steady-state reaction rate. A lag phase, whic h was NGD(+) concentration dependent, was also observed. The burst siz e indicates that the dimeric enzyme has a single catalytic site formed by two subunits, Pre-steady-state quench experiments did not detect c ovalent intermediates. Nicotinamide hydrolysis of NGD(+) precedes cycl ization and the chemical quench decomposes the enzyme-bound species to a mixture of cyclic and hydrolysis products. The time dependence of t his ratio indicated that nicotinamide bond-breakage occurs 4 times fas ter than the conversion of the intermediate to products. Product relea se is the overall rate-limiting step for enzyme reaction with NGD(+).