Biochemistry 1998, 37, 13239-13249
13239
The Reaction Mechanism for CD38. A Single Intermediate Is Responsible for Cyclization, Hydrolysis, and Base-Exchange Chemistries† Anthony A. Sauve,‡ Cyrus Munshi,§ Hon Cheung Lee,§ and Vern L. Schramm*,‡ Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, and Department of Physiology, UniVersity of Minnesota, Minneapolis, Minnesota 55455 ReceiVed May 27, 1998
ABSTRACT: 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-7deaza-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 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+.
Human CD38 is a glycoprotein expressed during early and late stage maturation in the plasma membrane of β cells (14). Reports link its presence to cytokine-induced differentiation (1, 5-7), cell adhesion (1, 6, 8) and signal transduction (5-13). A catalytic function in vivo has been proposed from the high sequence homology (68%) between CD38 and ADPribosyl-cyclase1 (14). ADP-ribosyl cyclase converts NAD+ to cyclic ADP-ribose (cADPR) (15, 16). cADPR is a potent agonist of calcium release from intracellular Ca2+ stores and † This work was supported by NIH Postdoctoral Fellowship GM19335 and Research Grant AI34342 from the NIH. * To whom correspondence should be addressed: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461. Telephone: (718) 430-2813. FAX: (718) 430-8565. E-mail: vern@ aecom.yu.edu. ‡ Albert Einstein College of Medicine. § University of Minnesota. 1 Abbreviations: ADP-ribosyl, adenosine diphosphate-ribosyl; ADPR, adenosine diphosphate-ribose; cADPR, cyclic adenosine diphosphateribose; GDPR, guanosine diphosphate-ribose, cGDPR, cyclic guanosine diphosphate-ribose; NAD+, nicotinamide diphosphate-ribose (oxidized form); NMN+, nicotinamide mononucleotide; NGD+, nicotinamideguanine dinucleotide; NHD+, nicotinamide-hypoxanthine dinucleotide; 7-deaza-NHD+, nicotinamide-7-deaza-hypoxanthine dinucleotide; TFA, trifluoroacetic acid; TCA, trichloroacetic acid.
acts as a second messenger in a signal cascade from NO synthesis to the release of calcium caused by cADPR binding to ryanodine receptors (1, 16-22). Both ADP-ribosyl cyclase and CD38 possess NAD+ cyclase activity (11). CD38 has been implicated in the regulation of cADPR levels in mammalian cells (20-22). Moreover, cADPR appears to increase Ca2+ levels in specific cell types (20-22). The wide distribution of CD38 in brain (20-24), white blood cells (2), pancreas (25), and a variety of other tissues (4, 26, 27) suggests that this molecule may have a general signaling role via cADPR production in vivo. The crystal structure of unliganded ADP-ribosyl cyclase from Aplysia reveals a homo-dimer (28). Each subunit possesses a cleft capable of accommodating NAD+. In the cavity between the subunits a dinucleotide can be modeled in the geometry for intramolecular cyclization. It has been suggested that CD38 is a dimer with one active site for cADPR formation and a second for cADPR hydrolysis (28). Recombinant CD38 lacking the membrane-anchoring Nterminal domain has shown that the enzymatic activity of CD38 resides wholly in the cloned ecto-cellular domain (29, 30). The availability of recombinant CD38 facilitates the determination of the reaction mechanism.
S0006-2960(98)01248-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 08/29/1998
13240 Biochemistry, Vol. 37, No. 38, 1998 Two mechanisms have been proposed to explain the reactivity of NAD+ and other dinucleotides with CD38 (11, 31-35). One mechanism proposes an obligatory cyclization of NAD+ to cADPR followed by hydrolysis to ADP-ribose (ADPR) (11). The second proposal is based on the mechanism of spleen NAD+ glycohydrolases which also function as NAD+ and NGD+ cyclases and hydrolases (32-35). The mechanism of NAD+ glycohydrolases and that suggested for CD38 proposes that all products are derived from a single intermediate that partitions to the cyclic or hydrolytic products. The present work resolves these views of the enzymatic mechanism of CD38. Spectroscopic, chemical reactivity, kinetic, and rapid quench studies are presented to furnish a complete microscopic analysis of the catalytic and rate steps for CD38. METHODS AND MATERIALS General Experimental. Solvents, compounds, and enzymes were used as obtained from commercial vendors unless otherwise indicated. Adenosine kinase was purified from beef liver (36). The stopped-flow spectrophotometer was an Applied Photophysics Model SX 17MV interfaced to an Acorn computer processing system using AP software. The rapid-quench instrument was a Kintek Model RQF-3 fitted with eight variable volume loops. HPLC used a Hitachi L-6200 combined with a digital variable wavelength LINEAR UVIS 204 detector set to 260 nm and a Hitachi D-2000 recorder/integrator. Recombinant CD38 was made and purified according to published procedures (30). Synthesis of 5′-Phospho-7-deaza-inosine. To a suspension of 2.0 mg (7.5 µmol) of tubercidin in 1.5 mL of 50 mM K2PO4 at pH 7.5 was added 50 µL of adenosine deaminase (calf intestine, 10 mg/mL) obtained from Boehringer Mannheim. The reaction was incubated at 37 °C for 12 h after which an additional 50 µL of enzyme was added. Progress of the reaction to the product 7-deaza-inosine was monitored by HPLC using a C-18 Waters Bondapak column (2.5 mm × 100 mm) eluted with: 2 mL/min 96:4 A/B (A, 50 mM ammonium acetate, pH 5.0; B, 50% H2O, 50% MeOH). The retention times were 24.0 min for tubercidin and 19 min for 7-deaza-inosine. After conversion in excess of 98%, the reaction was terminated by heating for 1 min at 110 °C and spinning for 2 min by tabletop centrifugation (12 000 min-1) to pellet protein. To the decanted supernatant was added an equal volume of 100 mM PIPES pH 6.5 buffer containing 2 mM ATP, 10 mM phosphoenol pyruvate, 100 mM KCl, and 10 mM MgCl2. Adenosine kinase and pyruvate kinase (0.5 unit each) were added to start the reaction. The reaction was incubated at 37 °C for 3.5 h until the reaction was in excess of 90% complete as determined by HPLC (see below). The product 5′-phospho-7-deaza-inosine was purified on a Waters C-18 Bondapak column using an ion-pairing solvent system 85:15 A/B (A, 100 mM triethylammonium acetate, pH 6.0; B, 50% MeOH, 50% H2O) with an elution time of 5.0 min. The phosphate compound was lyophilized and repurified by HPLC elution 95:5 A/B (A, 0.1% TFA; B, 50% MeOH, 50% H2O) and lyophilized. Treatment of this product with alkaline phosphatase rapidly regenerated the 7-deaza-inosine. Synthesis of 7-Deaza-nicotinamide Hypoxanthine Dinucleotide. The monophosphate was coupled to repurified NMN+
Sauve et al. (Sigma, TFA conditions for repurification as described above) by a known procedure (37, 38). The 7-deaza nucleotide and NMN+ in respective amounts 2.5 µmol and 5 µmol and 60 µmol of MgCl2 in 100 µL of distilled H2O were evaporated by tabletop spin evaporator to dryness; 100 µL of 1.5 M HEPES-NaOH and 100 µL 5 M of 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added to the dried residue to initiate the coupling reaction. Incubation (15 h) at 37 °C was followed by dilution with 1.0 mL of water. The reaction mixture was purified by injection of 500 µL onto a Waters C-18 Bondapak column 2.5 mm × 100 mm using elution of 95:5 A/B (A, 100 mM triethylammonium acetate, pH 6.0; B, 50% MeOH, 50% H2O). Lyophilization of the collected fractions afforded a 46% yield of 7-deaza-NHD+. Chemical hydrolysis at 80 °C of this compound yielded nicotinamide and a second compound assigned to 7-deaza-inosine diphosphate-ribose. Cleavage of the diphosphate bond of 7-deaza-NHD+ with snake venom diesterase provided NMN+ and 5′-phospho7-deaza-inosine (38). Synthesis of [1′-3H]NGD+ and [8-3H,carbonyl-14C]NGD+. [1′-3H]NAD+ was prepared as reported (39) and was treated with snake venom diesterase to obtain [1′-3H]NMN+ (39). The reaction mixture was heated to 110 °C for 40 s and spun to pellet protein; the supernatant was injected onto a C-18 column and eluted with 98:2 A/B (A, 0.1% TFA; B, 50% MeOH, 50% H2O). The peak containing [1′-3H]NMN+ was collected and lyophilized. The [1′-3H]NMN+ residue was diluted to a concentration of 2 mM with 50 mM potassium phosphate pH 7.5 containing 5 mM MgCl2 and 4 mM GTP. To this solution was added 1.0 unit of NAD+ pyrophosphorylase and 0.1 unit of inorganic pyrophosphatase. The reaction was incubated at 37 °C and after 6 h 1.0 unit of NAD+ pyrophosphorylase and 0.1 unit alkaline phosphatase were added and the reaction was continued for another 6 h. The reaction mixture was terminated by heating for 20 s at 110 °C to precipitate protein and then spun at 12 000 min-1 for 2 min to remove precipitate. The product [1′-3H]NGD+ was purified by injection of 500 µL onto a preparative C-18 column eluted with 50 mM ammonium acetate, pH 5.0. Eluted fractions of [1′-3H]NGD+ were lyophilized and were compared with authentic NGD+ (Sigma) to verify purity. [8-3H]NGD+ was synthesized as above by incubating [8-3H]GTP with NMN+ and coupling with NAD+ pyrophosphorylase. After 12 h of coupling as above, the product [8-3H]NGD+ was purified. The isolated [8-3H]NGD+ was incubated in the presence of 20 mM [carbonyl-14C]nicotinamide in 50 mM potassium phosphate pH 7.5. CD38 was added to a concentration of 0.05 µM, and the reaction monitored for incorporation of 14C into NGD+ by HPLC and scintillation counting. After the specific activity of 14C in NGD+ reached a plateau, the reaction was terminated by heating to 110 °C for 1 min and the reaction spun for 2 min to precipitate protein. The reaction was injected onto an HPLC column and the [8-3H, carbonyl-14C]NGD+ purified as above. Specific activity of [8-3H, carbonyl-14C]NGD+ was determined to be 30 000 cpm /nmol,3H cpm /14C cpm ) 0.58 Kinetic Study of NMN+ Hydrolysis by CD38. The desired concentrations of NMN+ in 50 mM potassium phosphate pH 7.5 were prepared to volumes of 200 µL and equilibrated at 37 °C; 1 µL of 380 nM CD38 was added to start reactions.
Reaction Mechanism of CD38 Aliquots were taken at 30 s time intervals and quenched with a 10 vol % solution of 25% TCA. Assay was done by HPLC 95:5 A/B (A, 50 mM ammonium acetate, pH 5.0; B, 50% MeOH, 50% H2O) on a Waters C-18 column to determine the amount of NMN+ unreacted and the amount of nicotinamide formed. Plots of product formed vs time were used to determine initial reaction rates. A plot of NMN+ concentration vs rate was fit using the program Kaleidograph to obtain the Michaelis parameters. Enzymatic Assay of 7-Deaza-NHD+and NHD+. Solutions of 7-deaza NHD+ and NHD+ from 10 to 400 µM in 100 µL of 50 mM potassium phosphate pH 7.5 were equilibrated at 37 °C. 5 µL of CD38 (830 nM) was added to start each reaction. Aliquots (20 µL) of the reaction were taken at 30 s intervals and quenched with a 10 vol % solution of 25% TCA. Injection of samples onto a Waters C-18 column eluted with a 98:2 A/B (A, 50 mM ammonium acetate; B, 50% MeOH, 50% H2O) at 2.0 mL/min permitted quantitation of the reaction components. Product formed vs time was plotted to determine initial reaction rates. The rates were plotted against substrate concentration and fit with Kaleidograph to a Michaelis curve to determine values for kcat and Km. NGD+ at saturating concentrations (>50 µM) was run as a control. Methanolysis of cADPR and NAD+ by CD38. Solutions of 500 µM NAD+ and 300 µM cADPR in 50 mM potassium phosphate pH 7.5 containing MeOH concentrations of 0.5, 1.0, and 2.0 M were prepared in volumes of 100-130 µL. CD38 was added (5.0 µL of 1.6 µM) to start the reaction and samples were taken to approximately 80% completion of reaction as determined by HPLC 96:4 A/B (A, 50 mM ammonium acetate, pH 5.0; B, 50% MeOH, 50% H2O). The amounts of ADPR and R- and β-methoxy-ADPR were quantitated by HPLC peak area measurements. A control reaction without enzyme was used for each substrate in MeOH /H2O 10:90 at pH 7.5 in 50 mM potassium phosphate buffer. Ratios of methoxy ethers to ADPR were determined by the ratio of peak areas. Peak areas were also used to determine the total amount of products formed of each species. Each reaction condition was plotted as follows: RM/H (RM/H ) moles of methanolysis product/moles of hydrolysis product) vs MeOH concentration. Relative nucleophilicity was calculated by the equation RN ) RM/H /([MeOH]/[H2O]) where [H2O] ≈ 55 M. Stopped-Flow Studies of NGD+ Cyclization. Fluorescence spectroscopy experiments were performed using a stoppedflow spectrophotometer fitted with dual syringe mounts and each contributing approximately 20 µL to give the total reaction volume of 40 µL. To the first syringe was added a CD38 solution of 4.0 or 8.0 µM in 50 mM acetate and 100 mM NaCl at pH 5.0. The other syringe contained 15 µM or 75 µM NGD+ in 50 mM potassium phosphate pH 7.5. Excitation wavelength was 300 nm and emission intensity was observed using a 360-nm cutoff filter. Data were collected at time resolutions to 0.25 ms. The dead time on the instrument is
