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

Biochemistry. 1998 Sep 22;37(38):13239-49. doi: 10.1021/bi981248s.


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 kcat 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 beta-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+.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ADP-ribosyl Cyclase
  • ADP-ribosyl Cyclase 1
  • Adenosine Diphosphate Ribose / analogs & derivatives*
  • Adenosine Diphosphate Ribose / chemistry
  • Antigens, CD*
  • Antigens, Differentiation / chemistry*
  • Binding, Competitive
  • Catalysis
  • Cyclic ADP-Ribose
  • Fluorescence Polarization
  • Guanosine Diphosphate Sugars / chemistry*
  • Humans
  • Hydrolysis
  • Kinetics
  • Membrane Glycoproteins
  • Methanol
  • NAD / analogs & derivatives
  • NAD / chemistry
  • NAD+ Nucleosidase / chemistry*
  • Niacinamide / pharmacology
  • Nicotinamide Mononucleotide / chemistry
  • Spectrometry, Fluorescence
  • Substrate Specificity


  • Antigens, CD
  • Antigens, Differentiation
  • Guanosine Diphosphate Sugars
  • Membrane Glycoproteins
  • cyclic guanosine diphosphate-ribose
  • NAD
  • Nicotinamide Mononucleotide
  • Cyclic ADP-Ribose
  • Adenosine Diphosphate Ribose
  • nicotinamide-hypoxanthine dinucleotide
  • Niacinamide
  • ADP-ribosyl Cyclase
  • CD38 protein, human
  • NAD+ Nucleosidase
  • ADP-ribosyl Cyclase 1
  • Methanol