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 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+.
Sauve
A A
AA
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Munshi
C
C
Lee
H C
HC
Schramm
V L
VL
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives* / Q000737:chemistry; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000737:chemistry*; D001667:Binding, Competitive; D002384:Catalysis; D036563:Cyclic ADP-Ribose; D005454:Fluorescence Polarization; D006156:Guanosine Diphosphate Sugars / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008562:Membrane Glycoproteins; D000432:Methanol; D009243:NAD / Q000031:analogs & derivatives / Q000737:chemistry; D009244:NAD+ Nucleosidase / Q000737:chemistry*; D009536:Niacinamide / Q000494:pharmacology; D009537:Nicotinamide Mononucleotide / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D015703:Antigens, CD; D000943:Antigens, Differentiation; D006156:Guanosine Diphosphate Sugars; D008562:Membrane Glycoproteins; C090657:cyclic guanosine diphosphate-ribose; D009243:NAD; D009537:Nicotinamide Mononucleotide; D036563:Cyclic ADP-Ribose; D000246:Adenosine Diphosphate Ribose; C032140:nicotinamide-hypoxanthine dinucleotide; D009536:Niacinamide; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1; D000432:Methanol