Identification of an unusual AT(D)Pase-like activity in multifunctional NAD glycohydrolase from the venom of Agkistrodon acutus

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Biochimie 91 (2009) 240e251 www.elsevier.com/locate/biochi

Research paper

Identification of an unusual AT(D)Pase-like activity in multifunctional NAD glycohydrolase from the venom of Agkistrodon acutus Liyun Zhang a, Xiaolong Xu a,*, Zhaofeng Luo b, Dengke Shen a, Hao Wu a a

Department of Chemistry, University of Science and Technology of China, No. 96, Jinzhai Road, Hefei, Anhui 230026, PR China b School of Lifesciences, University of Science and Technology of China, No. 96, Jinzhai Road, Hefei, Anhui 230026, PR China Received 15 April 2008; accepted 24 September 2008 Available online 9 October 2008

Abstract NAD-glycohydrolases (NADases) are ubiquitous enzymes that possess NAD glycohydrolase, ADPR cyclase or cADPR hydrolase activity. All these activities are attributed to the NADase-catalyzed cleavage of CeN glycosyl bond. AA-NADase purified from the venom of Agkistrodon acutus is different from the known NADases, for it consists of two chains linked with disulfide-bond(s) and contains one Cu2þ ion. Here, we show that AA-NADase is not only able to cleave the CeN glycosyl bond of NAD to produce ADPR and nicotinamide, but also able to cleave the phosphoanhydride linkages of ATP, ADP and AMP-PNP to yield AMP. AA-NADase selectively cleaves the PeOeP bond of ATP, ADP and AMP-PNP without the cleavage of PeOeP bond of NAD. The hydrolysis reactions of NAD, ATP and ADP catalyzed by AA-NADase are mutually competitive. ATP is the excellent substrate for AA-NADase with the highest specificity constant kcat/Km of 293  7 mM1 s1. AA-NADase catalyzes the hydrolysis of ATP to produce AMP with an intermediate ADP. AA-NADase binds with one AMP with high affinity determined by isothermal titration calorimetry (ITC). AMP is an efficient inhibitor against NAD. AA-NADase has so far been identified as the first unique multicatalytic enzyme with both NADase and AT(D)Pase-like activities. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: AA-NADase; Agkistrodon acutus; AT(D)Pase-like activity; NAD; ATP; ADP

1. Introduction The ubiquitous NAD-glycohydrolases (NADases, EC 3.2.2.5) that were first identified in 1942 [1] have been described for a wide range of prokaryotic and eukaryotic systems [2,3]. Generally, prokaryotes possess soluble NADases, whereas eukaryotes possess membrane-associated NADases [2,4]. These Abbreviations: NAD, nicotinamide adenine dinucleotide; NADases, NADglycohydrolases; AA-NADase, NADase from the venom of Agkistrodon acutus; apo-AA-NADase, Cu2þ-free AA-NADase; AHB-NADase, NADase from the venom of Agkistrodon hallys Blomhoffi; BF-NADase, NADase from the venom of Bungarus fasciatus; sNADase, a solubilized form of porcine brain NADase; ITC, isothermal titration calorimetry; ADPR, adenosine diphosphate ribose; cADPR, cyclic ADP-ribose; ADPRCs, ADP-ribosyl cyclases; NGD, nicotinamide guanine dinucleotide; GDPR, GDP-ribose; cGDPR, cyclic GDP-ribose; AMP-PNP, adenosine 50 -(b,g-imido) triphosphate. * Corresponding author. Tel.: þ86 551 3603214; fax: þ86 551 3603388. E-mail address: [email protected] (X. Xu). 0300-9084/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2008.09.003

enzymes catalyze the hydrolysis of nicotinamide adenine dinucleotide (NADþ) at the nicotinamideeribose linkage to yield adenosine diphosphate ribose (ADPR) and nicotinamide. Most eukaryotic NADases are bifunctional and can catalyze both the synthesis and hydrolysis of cyclic adenosine diphosphate ribose (cADPR), a second messenger that can mobilize Ca2þ from intracellular stores and regulate Ca2þ signaling [5,6]. These NADases that contain an ADP-ribosyl cyclase activity belong to the family of ADP-ribosyl cyclases (ADPRCs). Unlike eukaryotic NADases, most prokaryotic enzymes that belong to this group are unable to catalyze the synthesis and hydrolysis of cADPR. In view of the multifunction of NADases involved in catalyzing the hydrolysis of NAD to produce ADPR, as well as in metabolizing Ca2þ messenger cADPR, these enzymes from both prokaryotic and eukaryotic sources have received considerable attention in recent years. Numerous NADases and ADPRCs have been described from mammals and humans, such as bovine liver mitochondrial NADþ glycohydrolase [5], bovine spleen NADþ

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glycohydrolase [7], CD38 [8], and CD157 [9], among which, CD38 is the most extensively studied enzyme [10e18]. According to Lee, these NADases that contain an ADP-ribosyl cyclase activity are also referred to as CD38-like enzyme [8]. Snake venoms have been found to contain many kinds of proteins and enzymes. At present, several NADases, such as AHB-NADase, BF-NADase and AA-NADase, have been purified from the venoms of Agkistrodon hallys Blomhoffi [19], Bungarus fasciatus [20] and Agkistrodon acutus [21], respectively. In contrast to mammalian NADases, the purified snake venom enzyme is very stable. Reactions catalyzed by the snake venom NADases, therefore, represent an ideal experimental system for mechanistic studies of ADP-ribose transfer reactions. All reported NADases from snake venoms were characterized as catalyzing the hydrolysis of the nicotinamideeribose bond of NAD to produce ADPR and nicotinamide. It remains unclear whether the snake venom NADases also contain an ADP-ribosyl cyclase activity. AA-NADase was first purified from the venom of A. acutus by Huang et al. [21]. Our previous studies showed that AA-NADase consisted of two chains linked with disulfidebond(s) with a molecular weight of 100 kDa under nonreducing conditions and 50 kDa under reducing conditions [22,23]. Especially, among the known NADases, only AA-NADase contained Cu2þ ions that were essential for catalyzing the hydrolysis of NAD and nicotinamide guanine dinucleotide (NGD) [21]. Electron paramagnetic resonance spectroscopy revealed that the Cu2þ ion in AA-NADase might be coordinated with three N atoms and one water molecule [22]. Previous studies have shown that all known NADases including snake venom NADases only selectively cleave the Ce N glycosyl bond, except for sNADase, a solubilized form of porcine brain NADase [24]. sNADase has been found to selectively cleave the PeO bond of the adenosine side of NAD without the hydrolysis of the nicotinamideeribose pyridinium linkage. Interestingly, ATP has been reported to inhibit the cADPR-hydrolyzing activity of CD38, resulting in the increased formation of cADPR [25]. The initial aim of this study was to investigate whether AA-NADase catalyzed the synthesis of cADPR from NAD and whether ATP inhibited the NADase activity of AA-NADase. Surprisingly, the present results indicate that AA-NADase is not only able to cleave the CeN glycosyl bond of NAD to produce ADPR and nicotinamide, but also able to cleave the PeOeP bond of ATP, ADP and AMPPNP to produce AMP. Furthermore, the hydrolyses of NAD, ATP and ADP catalyzed by AA-NADase are mutually competitive. AA-NADase is so far identified as the first unique multicatalytic enzyme with both NADase and AT(D)Pase-like activities. The unusual enzymatic activity of AA-NADase opens a new area of investigation into the mechanism of its action involved in energy metabolism, signal transduction, ageing, and cellular injury.

The standard reaction mixture that contained 20 mM Trise HCl, pH 7.4, 1.0 mg AA-NADase, 0.5 mM adenosine derivative (ATP, ADP or AMP) and 0.1e1.0 mM NAD in a total volume of 2 ml was incubated at 37  C. At timed intervals, 10 ml aliquots of the reaction mixture were analyzed by HPLC.

2. Materials and methods

2.5. Assays for AT(D)Pase-like activity

2.1. Chemicals

Reaction mixtures were maintained at 37  C and contained 20 mM TriseHCl, pH 7.4, 1.0 mg AA-NADase and 0.5 mM substrate (ATP, ADP, AMP or AMP-PNP), in a final volume of 2.0 ml. At timed intervals, 10 ml aliquots were immediately

Lyophilized venom powder was provided by the TUN-XI Snakebite Institute (AnHui, PR China). ATP, ADP, and AMP,

being ultra pure grades (98%), were purchased from Amresco. b-NAD (98%) was obtained from Roche. The products for high performance liquid chromatography were obtained from Shimadzu. All other reagents were of analytical reagent grade. Milli-Q purified water was used throughout. 2.2. Purification of AA-NADase The purification of AA-NADase was performed as previously described [23]. Protein purity was confirmed using SDSePAGE analysis and the concentration of AA-NADase was calculated from the absorption coefficient (A1% 1 cm ¼ 0.66) at 280 nm and the relative molecular weight (Mr ¼ 100 kDa). The apo-AA-NADase was prepared by dialysis of purified AA-NADase against 2 mM EDTA in 20 mM TriseHCl (pH 7.4) for 24 h and then extensively against 20 mM TriseHCl (pH 7.4). 2.3. Assays for NAD glycohydrolase activity The NADase activity was assayed by high performance liquid chromatography. Reaction mixtures were maintained at 37  C and contained 20 mM TriseHCl buffer, pH 7.4, 0.5e 1.0 mg AA-NADase and 0.1e1.0 mM NAD, in a final volume of 2.0 ml. At timed intervals, 10 ml aliquots were immediately injected into the HPLC column using a 10-ml sample syringe. High performance liquid chromatography of products was performed on an LC-20AD (Shimadzu, Japan) HPLC equipped with an SPD-M20A spectrophotometric detector. Separations were made on a Shim-pack VP-ODS (250  4.6 mm) column. The mobile phase contained 10 mM ammonium phosphate, pH 5.5, acetonitrile (100:2 v/v). Product concentrations were calculated by an LC-20AD integrator previously calibrated with known concentrations of the products to be analyzed. The kinetic experiment for the hydrolysis of NAD by AA-NADase was done at increasing concentrations of NAD (0.1e1.0 mM). For each substrate concentration, the initial rate was calculated from the linear region of the progress curve for the dependence of the substrate content on incubation time. The kinetic parameters were obtained from the plot of the initial rates as a function of substrate concentrations, using a nonlinear regression program. 2.4. Inhibition of AA-NADase by adenosine derivative

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injected into the HPLC column. The kinetic experiments for the hydrolysis of ATP, ADP and AMP-PNP by AA-NADase were done at increasing concentrations (0.1e1.0 mM) of ATP, ADP or AMP-PNP. 2.6. Isothermal titration calorimetry (ITC) measurements Isothermal titration calorimetry experiments were carried out at 25  C on a MicroCal VP-ITC microcalorimeter (MicroCal, LLC), as described by Todd and Gomez [26]. All buffers and solutions were degassed immediately before each titration. The reference cell was filled with buffer. The sample cell was filled with 5 mM AA-NADase and 20 mM TriseHCl, pH 7.4, and the titration syringe was filled with 0.5 mM AMP solution in 20 mM TriseHCl, pH 7.4. The system was equilibrated at 25  0.1  C with stirring at 307 rpm prior to the addition of 240 ml of 0.5 mM AMP solution. The protein in the 1.468 ml calorimeter cell was titrated by AMP solution by 30 successive automatic injections of 8 ml each. Before the termination of the experiment, the thermal power was allowed to return to the level matching the original baseline, indicating complete reaction. The area under the baseline was integrated and divided by the total amount of AMP added to the cell to determine the molar enthalpy of the reaction [27]. Thermodynamic parameters N (stoichiometry), KA (association constant), and DH (enthalpy change) were obtained by nonlinear least-squares fitting of the experimental data using a model of one site of the Origin software package (version 7.0) provided with the instrument. The free energy of binding (DG) and entropy change (DS ) were obtained using the following equations. DG ¼ RT ln KA

ð1Þ

DG ¼ DH  TDS

ð2Þ

The affinity of AMP to protein was given as the dissociation constant (KD ¼ 1/KA). For each proteineAMP interaction, three titrations were performed. Titration data were analyzed independently, and the thermodynamic values obtained were averaged. 3. Results 3.1. Hydrolysis of NAD by AA-NADase AA-NADase has been purified from the crude venom powder by a three-step chromatography procedure [23]. As shown in Fig. 1, AA-NADase gives a single band corresponding to 100 kDa before reduction and gives a single band corresponding to 50 kDa after reduction, which clearly shows that the purified AA-NADase is fairly homogeneous and consists of two chains connected by disulfide linkages, as reported previously [23]. Our recent results show that AANADase is able to catalyze the cleavage of the nicotinamidee ribose linkage of nicotinamide guanine dinucleotide (NGD), an NAD analogue, to produce cyclic GDP-ribose (cGDPR) and GDP-ribose (GDPR) (unpublished data). In order to

Fig. 1. SDS-polyacrylamide gel electrophoresis of AA-NADase. Purified AANADase was electrophoresed in 10% polyacrylamide gels containing 0.1% SDS under nonreducing (NR) and reducing (R) conditions with 0.1 M DTT. Lane 1 and lane 3, AA-NADase; lane 2, molecular mass markers.

investigate whether AA-NADase also catalyzes the synthesis of cADPR from NAD, we studied the AA-NADase-catalyzed reaction of NAD by HPLC. As shown in Fig. 2, when NAD was incubated in the presence of AA-NADase, two peaks appeared at a retention time of 6.8 min for ADPR and 3.3 min for nicotinamide, and the peak of NAD with a retention time of 8.7 min decreased in time-dependence. The kinetic parameters for the hydrolysis of NAD by AA-NADase were determined from the reaction rates monitored on the HPLC profiles. Fig. 2C shows the typical LineweavereBurk plot of the data. A mean Km of 0.26  0.03 mM, a mean maximum reaction rate (Vmax) of 6.7  0.2 mM min1 and a mean catalytic constant (kcat) of 22.3  0.5 (s1) for the hydrolysis of NAD catalyzed by AA-NADase were obtained from three independent experiments. No peak was detected for cADPR as monitored by HPLC (Fig. 2A). 3.2. Effect of Cu2þ on the NADase activity of AANADase Among the known NADases, only AA-NADase contains copper ions. Huang et al. have shown that copper ions are essential for the NADase activity of AA-NADase determined by fluorescence assay and that the binding of Cu2þ ions to AA-NADase is not reversible [21]. Here we have re-examined the effect of Cu2þ on its NADase activity by HPLC. As shown in Fig. 3A and B, AA-NADase completely loses its NADase activity after the removal of copper ions from the protein. The observation is in agreement with the result of Huang et al. [21] and further confirms that copper ions are essential for the NADase activity of AA-NADase. As shown in Fig. 3C and D, however, apo-AA-NADase can recover 73% of its NADase activity after addition of 10 mM Cu2þ. In control experiments (without apo-AA-NADase), the peak of NAD does not change and no new peak appears after 60 min of incubation of 0.5 mM NAD with 10 mM Cu2þ in the same buffer, suggesting that

L. Zhang et al. / Biochimie 91 (2009) 240e251

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B

1.0

ADPR

Relative Content

nicotinamide

Inc ub ati (m on ti in) me

NAD

A

120 45 30 15 0 0

0.8

NAD

0.6

ADPR

0.4

nicotinamide 0.2 0.0

2

4

6

8

10

12

14

16

0

Retention time (min)

20

0.56

60

80

100

120

C

0.48

1/V0 ( min.µM-1 )

40

Incubation time (min)

0.40 0.32 0.24 0.16 0

2

4

6

8

10

12

1/NAD (mM)-1 Fig. 2. Hydrolysis of NAD by AA-NADase. (A) NAD (0.5 mM) was incubated with 1 mg AA-NADase in 20 mM TriseHCl (pH 7.4) at 37  C in a final volume of 2.0 ml. HPLC assay as described under Section 2. (B) Changes in NAD, ADPR, and nicotinamide content are plotted as functions of incubation time. (C) LineweavereBurk plot of the hydrolysis of NAD by AA-NADase. The data represent the means of three experiments.

2

4

6

8

10

12

cu In

Relative Content

ba t (m ion in tim ) e

A

50 37.5 25 12.5 0 0

B

1.4

NAD

1.2 1.0 0.8 0.6

14

Retention time (min)

0

10

20

30

40

50

Incubation time (min) ADPR

In cu

ba (m tion in ti m )

e

NAD

C

45 30 15 0

0

2

4

6

8

10

12

Retention time (min)

14

Relative Content

0.8

nicotinamide

NAD

D

0.6 0.4 0.2

ADPR nicotinamide

0.0 0

10

20

30

40

50

Incubation time (min)

Fig. 3. Effect of Cu2þ on the NADase activity of AA-NADase. (A) NAD (0.5 mM) was incubated with 0.5 mg apo-AA-NADase in 20 mM TriseHCl (pH 7.4) at 37  C in a final volume of 2.0 ml. The products were analyzed by HPLC as described under Section 2. (B) Change in NAD content is plotted as functions of incubation time for the reaction in panel (A). (C) NAD (0.5 mM) was incubated with 0.5 mg apo-AA-NADase in 20 mM TriseHCl (pH 7.4) in the presence of 10 mM Cu2þ at 37  C in a final volume of 2.0 ml. HPLC assay as described under Section 2. (D) Change in NAD, ADPR, and nicotinamide content are plotted as functions of incubation time for the reaction in panel (C). Values are means for three experiments.

L. Zhang et al. / Biochimie 91 (2009) 240e251

the hydrolysis of NAD in Fig. 3C is a specific AA-NADasecatalyzed reaction. This result demonstrates that the binding of copper ions to AA-NADase is reversible, which is contrary to the result of Huang et al. [21]. 3.3. Analysis of the inhibition of AA-NADase by ATP, ADP and AMP AA-NADase shows a cyclase activity to convert NGD to cyclic GDP-ribose, which encouraged us to examine the synthesis of cyclic ADP-ribose from NAD. However, the possible intermediate of cADPR cannot be detected by HPLC probably due to its low concentration. Akira Tohgo and coworkers reported that ATP inhibited the cADPR hydrolase activity of CD38, resulting in the increased formation of cADPR [28]. Whether ATP can inhibit the hydrolytic activity of AA-NADase was investigated by incubating the mixture of 0.5 mM ATP, 1.0 mg AA-NADase and 0.5 mM NAD and then

ADP

AMP NAD

e tim n io at in) 15 b cu (m 10 0

A

5

0

NAD

1.0

ATP

In

analyzing the reaction mixture by HPLC. As shown in Fig. 4A, a new peak on HPLC appears at a retention time of 7.1 min after incubation of NAD with AA-NADase in the presence of ATP. This new increasing peak is neither for ADPR nor for cADPR, because the peak for NAD does not change in the presence of ATP. Surprisingly, the intensity of the peak of ATP decreases in time-dependence with an initial reaction rate of 8.3  0.3 mM min1. From this result, we speculate that the unusual new peak should be a product of ATP hydrolysis, which may be ADP or AMP. After calibration of the column with ADP and AMP as standards, it has been found that the retention time of the new peak is equal to that of AMP, indicating that the new product is AMP. The results also show that the AA-NADase-catalyzed cleavage reaction of NAD is significantly inhibited in the presence of 0.5 mM ATP. Similarly, when NAD was incubated with AA-NADase in the presence of 0.5 mM ADP, a new increasing peak for AMP appears, while the ADP peak decreases with an initial reaction

Relative content

244

B

0.8

ATP

0.6 0.4

AMP 0.2

ADP

2

4

6

8

10

0.0

12

0

Retention time (min)

5

10

15

Incubation time (min)

AMP ADP

e

tim n it o ) ba in 90 cu (m 60

In

NAD

C

30 0

0

2

4

6

8

10

Relative content

1.2

ADP 0.6 0.4

AMP

0.2

Retention time (min)

0

20

40

60

AMP

AMP NAD

e tim n tio ) ba min 30 u ( 15 nc

E

0

Relative content

1.0

0

0.8

4

6

8

10

12

Retention time (min)

100

14

16

F

NAD

0.6 0.4 ADPR

0.2

nicotinamide

0.0 2

80

Incubation time (min)

nicotinamide ADPR

I

D

0.8

0.0

12

NAD

1.0

0

5

10

15

20

25

30

Incubation time (min)

Fig. 4. Inhibition of AA-NADase by ATP, ADP and AMP. The mixture of 0.5 mM NAD with 0.5 mM ATP (A), ADP (C) or AMP (E) was incubated with 1 mg AANADase in 20 mM TriseHCl (pH 7.4) at 37  C in a final volume of 2.0 ml. The products were analyzed by HPLC as described under Section 2. (B) Time course of changes in NAD, ATP, ADP, and AMP for the reaction in panel (A). (D) Time course of changes in NAD, ADP, and AMP for the reaction in panel (C). (F) Time course of changes in NAD, AMP, ADPR and nicotinamide for the reaction in panel (E). Values are means for three experiments. The column was calibrated with ATP, ADP, AMP as standards.

L. Zhang et al. / Biochimie 91 (2009) 240e251

rate of 2.6  0.1 mM min1 (Fig. 4C and D). The result shows that ADP is also hydrolyzed to form AMP in the reaction mixture and that AA-NADase-catalyzed cleavage reaction of NAD is also markedly inhibited in the presence of 0.5 mM ADP. Based on these observations, it seemed most likely that AA-NADase can catalyze the hydrolysis of adenosine derivatives ATP and ADP. As shown in Fig. 4E and F, when NAD was incubated with AA-NADase in the presence of 0.5 mM AMP, the intensity of NAD peak at a retention of 8.7 min slightly decreases while the intensity of nicotinamide peak at a retention of 3.3 min and the intensity of ADPR peak at a retention of 6.8 min slightly increase, revealing that the cleavage of NAD by AA-NADase is also markedly inhibited by 0.5 mM AMP. This result indicates that AMP is an efficient inhibitor for the NADase activity of AA-NADase. No obvious change has been observed for the AMP peak at a retention of 7.1 min, suggesting that AANADase can’t catalyze the hydrolysis of AMP. Taken together, these results show that all ATP, ADP and AMP inhibit the AA-NADase-catalyzed hydrolysis of NAD. Inhibition constants (Ki) for each adenosine derivative were determined at a constant adenosine derivative concentration while varying NAD concentration. Kinetic parameters for each inhibitory reaction were determined from the reaction rates monitored on the HPLC profiles. Fig. 5 clearly illustrates a competitive mode of inhibition for ATP, ADP and AMP with the Ki of 60.5  1.5 mM, 96.3  3.1 mM and 127  2.6 mM, respectively [29], indicating that the inhibitory capacity follows the trend ATP > ADP > AMP. 3.4. Hydrolysis of ATP, ADP and AMP by AA-NADase

1/V0 ( min.µM-1 )

Fig. 4 shows that both ATP and ADP are hydrolyzed in the presence of AA-NADase and NAD. To further confirm that it 4.0

NAD + ATP

3.2

NAD + ADP

2.4

NAD + AMP

1.6

0.8

-10

-5

NAD

0

5

10

15

20

1/NAD (mM)-1 Fig. 5. Competitive inhibition of AA-NADase by adenosine derivatives. The standard reaction mixture that contained 20 mM TriseHCl, pH 7.4, 0.6 mg AA-NADase, 0.5 mM adenosine derivatives (ATP, ADP or AMP) and 0.1e 1.0 mM NAD in a total volume of 2 ml was incubated at 37  C for 30 min. The products were analyzed by HPLC as described under Section 2. For each substrate concentration, the initial rate was calculated from the linear region of the progress curve for the dependence of the substrate (NAD) content on incubation time.

245

was AA-NADase that catalyzed the hydrolysis of ATP and ADP, we incubated 1.0 mg AA-NADase with 0.5 mM ATP, ADP or AMP in the absence of NAD. Fig. 6A and B shows that the peak of ATP decreases with an initial reaction rate of 9.6  0.3 mM min1, while the peak of AMP at a retention time of 7.1 min increases. In control experiments (without AA-NADase), no such peak appears at a retention time of 7.1 min. Fig. 6C shows that a small peak at a retention time of 4.6 min increases in 7.5 min and then reaches a near plateau from 7.5 min to 30 min and significantly decreases from 30 min to 60 min. The retention time of the small peak is identical to that of the internal ADP marker, indicating that it is the peak of ADP. Fig. 6D shows the typical Lineweavere Burk plot of the data. The Km of 0.14  0.01 mM, Vmax of 12.3  0.3 mM min1 and kcat of 41.0  0.8 (s1) were obtained from three independent experiments for the hydrolysis of ATP catalyzed by AA-NADase. In control experiments (without AA-NADase), the peak of ATP does not change and no new peak appears after 60 min of incubation of 0.5 mM ATP in the same buffer, suggesting that the hydrolysis of ATP is a specific AA-NADase-catalyzed reaction. As shown in Fig. 7A and B, similarly, the peak of ADP also decreases with an initial reaction rate of 2.9  0.1 mM min1, while the AMP peak at a retention time of 7.1 min increases. Fig. 7C shows the typical LineweavereBurk plot of the data. The Km of 0.06  0.01 mM, Vmax of 3.2  0.1 mM min1 and kcat of 10.7  0.2 (s1) were obtained from three independent experiments for the hydrolysis of ADP catalyzed by AANADase. In control experiments (without AA-NADase), the peak of ADP does not change and no new peak appears after 60 min of incubation of 0.5 mM ADP in the same buffer, suggesting that the hydrolysis of ADP is a specific AANADase-catalyzed reaction. All these observations taken together clearly indicate that AA-NADase indeed catalyzes the cleavage of both ATP and ADP to produce AMP. Therefore, AA-NADase is not only able to cleave the CeN glycosyl bond of NAD to produce ADPR and nicotinamide, but also able to cleave the PeOeP bond of both ATP and ADP to produce AMP. As shown in Fig. 8, when both substrates (0.5 mM ATP and 0.5 mM ADP) were simultaneously present, the peaks of both ATP and ADP decrease in time-dependence after incubation with 0.6 mg AA-NADase. The initial reaction rates of the hydrolysis of ATP and ADP to AMP catalyzed by AA-NADase were determined to be 2.8  0.1 mM min1 and 0.9  0.1 mM min1, respectively, from three independent experiments. This result indicates that when the two substrates (ATP and ADP) are present at a concentration ratio of 1:1, AA-NADase can catalyze the hydrolysis of both ATP and ADP with a higher hydrolysis rate for ATP than for ADP. From the values of the hydrolysis rates of ATP and ADP to AMP catalyzed by 0.6 mg AA-NADase, the hydrolysis rates of 0.5 mM ATP and 0.5 mM ADP to AMP catalyzed by 1.0 mg AA-NADase were estimated to be 4.7  0.2 mM min1 and 1.6  0.1 mM min1, respectively. The decreases in the hydrolysis rates of both ATP (from 9.6  0.3 mM min1 to 4.7  0.2 mM min1) and ADP (from 2.9  0.1 mM min1 to

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ATP

1.0

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60 30 22.5 15 7.5 0 0

2

Relative Content

In cu ba (m tion in ti ) m e

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ATP

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0.4 0.2

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Incubation time (min) 0.24

1/V0 ( min.µM-1 )

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ba (m tion in ti ) m e

ADP

C

60 30 22.5 15 7.5 0

D

0.20 0.16 0.12 0.08 0.04 0.00

4.4 4.6 4.8 5.0 5.2 5.4 5.6

0

Retention time (min)

2

4

6

8

10

12

1/ATP (mM)-1

Fig. 6. Hydrolysis of ATP by AA-NADase. (A) 1.0 mg of AA-NADase was incubated with 0.5 mM ATP in 20 mM TriseHCl (pH 7.4) at 37  C in a final volume of 2.0 ml. The products were analyzed by HPLC as described under Section 2. (B) Time course of changes in ATP, AMP and ADP for the reaction in panel (A). (C) Amplification of the peak with a retention time of 4.6 min in panel (A). (D) LineweavereBurk plot of the hydrolysis of ATP by AA-NADase. The data represent the means of three experiments.

B

Relative content

1.0

Inc ub at (m ion in) tim e

ADP AMP

A

45 30 15 0 0

ADP

0.8 0.6 0.4

AMP

0.2 0.0

2

4

6

8

10

12

0

14

15

30

45

Incubation time (min)

Retention time (min)

1/V0 ( min.µM-1 )

C 0.5

0.4

0.3 0

2

4

6

8

10

1/ADP (mM)-1 Fig. 7. Hydrolysis of ADP by AA-NADase. (A) 1 mg of AA-NADase was incubated with 0.5 mM ADP in 20 mM TriseHCl (pH 7.4) at 37  C in a final volume of 2.0 ml. The products were analyzed by HPLC as described under Section 2. (B) Time course of changes in ADP and AMP for the reaction in panel (A). (C) LineweavereBurk plot of the hydrolysis of ADP by AA-NADase. The data represent the means of three experiments.

L. Zhang et al. / Biochimie 91 (2009) 240e251

AMP

Inc ub

B

0.6

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ADP

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Fig. 8. Hydrolysis of ATP and ADP by AA-NADase. (A) A mixture of 0.5 mM ATP and 0.5 mM ADP was incubated with 0.6 mg AA-NADase in 20 mM TriseHCl (pH 7.4) at 37  C in a final volume of 2.0 ml. At the times indicated, 10 ml aliquots were withdrawn and analyzed by HPLC as described under Section 2. (B) Changes in ATP, ADP, and AMP content are plotted as functions of incubation time. Values are means for three experiments.

1.6  0.1 mM min1) in the presence of the two substrates suggest that the hydrolysis reactions of ATP and ADP catalyzed by AA-NADase are mutually competitive. As shown in Fig. 9, no obvious change has been observed for the peak of AMP after 30 min of incubation of AMP with AA-NADase, which further confirms that AMP is not a substrate of AA-NADase.

parameters for the hydrolysis of NAD, ATP, ADP and AMPPNP by AA-NADase are shown together in Table 1. In terms of efficiency (kcat/Km), among the four substrates (NAD, ATP, ADP and AMP-PNP), ATP is the best substrate for AA-NADase while AMP-PNP is the poorest substrate for AA-NADase. 3.6. Interaction of AA-NADase with AMP

3.5. Hydrolysis of AMP-PNP by AA-NADase In order to investigate whether AA-NADase catalyzes the hydrolysis of the ATP-analogue AMP-PNP that is nonhydrolysable by ATPases, we incubated 0.5 mM AMP-PNP with 1.0 mg AA-NADase. As shown in Fig. 10A, a new increasing peak for AMP at a retention time of 7.1 min appears, while the AMP-PNP peak at a retention time of 4.8 min decreases in time-dependence. The result shows that AA-NADase also catalyzes the hydrolysis of AMP-PNP to form AMP, indicating that AA-NADase is different from common ATPases. Fig. 10C shows the typical Lineweavere Burk plot of the data. A mean Km of 0.47  0.05 mM, a mean maximum reaction rate (Vmax) of 9.3  0.3 mM min1 and a mean catalytic constant (kcat) of 31.0  0.8 (s1) for the hydrolysis of AMP-PNP catalyzed by AA-NADase were obtained from three independent experiments. The kinetic

The inhibition of the NADase activity of AA-NADase by AMP suggests a possible binding between the protein and AMP. We utilized isothermal titration calorimetry (ITC) to test the binding between the protein and AMP. Fig. 11 shows a representative calorimetric titration of AA-NADase with AMP. The exothermic evolution of heat upon AMP injections shown in the upper panel illustrates AMP binding to the protein. The ITC data fitting indicates that the binding ratio between the protein and AMP is 0.92  0.27, which suggests one binding-site between the protein and AMP. The ITC data fitting also shows that the values of thermodynamic parameters, KD, DG, DH, and DS are 9.01  0.20 mM, 6.88  0.18 kcal mol 1, 5.39  0.48 kcal mol 1 , and 5.01  0.11 cal/(mol K), respectively. A comparison of DH and TDS shows that the binding interaction between AMP and AA-NADase is predominantly enthalpic in nature. These

B

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Fig. 9. Hydrolysis of AMP by AA-NADase. (A) 1 mg of AA-NADase was incubated with 0.5 mM AMP in 20 mM TriseHCl (pH 7.4) at 37  C in a final volume of 2.0 ml. The products were analyzed by HPLC as described under Section 2. (B) Time course of changes in AMP for the reaction in panel (A). Values are means for three experiments.

L. Zhang et al. / Biochimie 91 (2009) 240e251

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1/[AMP-PNP] (mM)-1 Fig. 10. Hydrolysis of AMP-PNP by AA-NADase. (A) 1.0 mg of AA-NADase was incubated with 0.5 mM AMP-PNP in 20 mM TriseHCl (pH 7.4) at 37  C in a final volume of 2.0 ml. The products were analyzed by HPLC as described under Section 2. (B) Time course of changes in AMP-PNP and AMP for the reaction in panel (A). (C) LineweavereBurk plot of the hydrolysis of AMP-PNP by AA-NADase. The data represent the means of three experiments.

results reveal that AMP indeed binds to AA-NADase with a large binding enthalpy in one high affinity site. 4. Discussion NADase is widely known to have NAD glycohydrolase, ADPR cyclase or cADPR hydrolase activity. All these activities are attributed to the NADase-catalyzed cleavage of CeN glycosyl bond. A new catalytic hydrolysis has been reported for sNADase that selectively cleaves the PeO bond of the adenosine side of NAD without the hydrolysis of the nicotinamideeribose pyridinium linkage [24]. One of the most intriguing observations in the present study is that AA-NADase possessing NADase activity is able to catalyze the unusual cleavage of the PeOeP bond of both ATP and ADP to produce AMP. Fig. 2 shows that AA-NADase has a typical NADase activity of ubiquitous NADases as reported by Huang et al. [21]. Figs. 6, 7 and 8 clearly show that AA-NADase has AT(D)Pase-like activity. To our Table 1 Kinetic parameters for hydrolysis of NAD, ATP, ADP and AMP-PNP by AANADase. The values are the mean  SD of n ¼ 3. Vmax (mM min1) kcat (s1)

Substrate

Km (mM)

NAD ATP ADP AMP-PNP

0.26  0.03 6.7  0.2 0.14  0.01 12.3  0.3 0.06  0.01 3.2  0.1 0.47  0.05 9.3  0.3

kcat/Km (mM1 s1)

22.3  0.5 86  3 41.0  0.8 293  7 10.7  0.2 178  4 31.0  0.8 66  5

knowledge, among the known NADases, none possesses this unusual cleavage activity. Therefore, AA-NADase has so far been identified as the first unique multicatalytic enzyme with both NAD glycohydrolase and AT(D)Pase-like activities. This observation also raises another question whether similar multicatalytic enzymes are distributed in the tissues of other animals than snakes. As shown in Figs. 4, 6 and 7, the initial hydrolysis rates of both ATP and ADP decrease from 9.6  0.3 mM min1 to 8.3  0.3 mM min1 and from 2.9  0.1 mM min1 to 2.6  0.1 mM min1, respectively, in the presence of 0.5 mM NAD, suggesting that both ATPase-like and ADPase-like activities are inhibited by NAD. On the other hand, as shown in Fig. 5, both ATP and ADP have been found to be competitive vs NAD. Therefore, the hydrolysis reactions of NAD, ATP and ADP catalyzed by AA-NADase are mutually competitive. It is highly possible that the two different reactions occur at a single active site with competitive inhibition. Further investigation is necessary to clarify this issue. Significant sequence homologies have been observed for known NADases [6,30e32]. The three-dimensional structures of Aplysia californica cyclase, CD38 and the ectodomain of CD157 have been found to be very similar [33e35]. They are all composed of two identical chains without disulfide linkage between two chains, and none of them contains metal ions. Contrary to these NADases, AA-NADase consists of two chains that are linked with disulfide-bond(s) [22,23]. Especially, AA-NADase contains Cu2þ ions [22,23]. The above results show that Cu2þ ions are essential for catalyzing the

L. Zhang et al. / Biochimie 91 (2009) 240e251

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Molar Ratio Fig. 11. Isothermal titration calorimetry (ITC) measurement of AMP binding to AA-NADase. (Upper panel) raw ITC data for injecting 0.5 mM AMP in 20 mM TriseHCl, pH 7.4 into 5 mM AA-NADase in the same buffer. (Lower panel) normalized ITC data for titrations plotted vs molar ratio of AMP/AANADase. Data analysis using Origin 7.5 software indicates that the binding data fit well to a model of one binding-site.

hydrolysis of NAD. Our recent result shows that each AANADase specifically binds with one Cu2þ ion and Cu2þ ion is also essential for catalyzing the hydrolysis of ATP and ADP (data not shown). Thus, both NAD glycohydrolase and AT(D)Pase-like activities of the protein depend on Cu2þ. To our knowledge, among the known NADases, only AANADase contains Cu2þ ion. However, Cu2þ ion has been reported to inhibit several ADPRCs, such as plasma membrane ADPR cyclase [36] and VSMC ADPR cyclase [37]. All these results taken together indicate that AA-NADase is quite different from common NADases, for it contains a Cu2þ and consists of two chains linked with disulfide-bond(s), which may be the reason why AA-NADase possesses the unique multicatalytic activity. Fig. 1 shows that the purified AA-NADase is fairly homogeneous, indicating that both NADase and AT(D)Pase-like activities should be contributed by the same protein, which is further supported by the facts that both NAD glycohydrolase and AT(D)Pase-like activities of the protein depend on Cu2þ and that the AA-NADase-catalyzed hydrolysis reactions of NAD, ATP and ADP are mutually competitive. Further identification of the multicatalytic activities of AA-NADase by cloning its cDNA and expressing the recombinant protein in a heterologous system is currently in progress.

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Fig. 2 shows that AA-NADase can catalyze the hydrolysis of the nicotinamideeribose bond of NAD to form ADPR and nicotinamide with a specificity constant kcat/Km of 86  3 mM1 s1, indicating that AA-NADase is an efficient protein for catalysis of NAD. However, no peak was observed for cADPR as monitored by HPLC under our experimental conditions. It has been reported that the cADPR/ADPR production ratio is 0.01e0.02 for most known NADases [38]. AA-NADase may also catalyze the synthesis of cyclic ADPribose from NAD, but the product of cADPR may be too scant to be detected by HPLC on account of its sensitivity limit. No peak has been observed in Fig. 2 for the product of the cleavage of the PeO bond of NAD, suggesting that unlike sNADase, AA-NADase cannot catalyze the cleavage of the PeO bond of NAD. As shown in Table 1, when considering NAD, ATP, ADP and AMP-PNP as competing substrates for the active site(s) of AA-NADase, the ratio of their specificity constants kcat/Km is in favor of ATP. It appears, therefore, that ATP is an excellent substrate for AA-NADase. Fig. 5 shows that all of ATP, ADP and AMP competitively inhibit the NADase activity of AA-NADase and their inhibitory capacity follows the trend ATP > ADP > AMP. A similar observation for calf spleen NADase was reported earlier by Schuber et al. [39]. ATP, ADP and AMP behaved as competitive inhibitors of calf spleen NADase, but calf spleen NADase could not hydrolyze ATP and ADP. Another early report showed that ATP, ADP and AMP did not inhibit the NADase activity of BF-NADase from the venom of B. fasciatus, even at concentrations up to 20 mM [40]. It is obvious from Fig. 8 that when the two substrates (ATP and ADP) are present, AA-NADase can catalyze the hydrolysis of both ATP and ADP. If AA-NADase catalyzes the hydrolysis of ATP to produce AMP without intermediate ADP, the ADP peak in the Fig. 6A and C should continuously decrease and could not reach a plateau in any incubation time. However, Fig. 6C shows that the peak of ADP increases first and then reaches a near plateau, suggesting that an intermediate of ADP is formed in the hydrolysis of ATP by AA-NADase. At the beginning of the incubation of the reaction mixture, the formation of ADP results in the increase of the ADP peak. After 60 min of incubation, in contrast, no ADP is formed as ATP has nearly been completely hydrolyzed, but the AA-NADase-catalyzed hydrolysis of ADP continues; as a result, the ADP peak in Fig. 6C decreases markedly. Fig. 4A also shows that the intensity of ADP peak does not change for up to 15 min, which further indicates that an intermediate of ADP is formed in the hydrolysis of ATP by AA-NADase. ATP contains two PeOeP phosphoanhydride bonds and ADP contains one PeOeP phosphoanhydride bond. The existence of intermediate ADP in the hydrolysis of ATP indicates that AA-NADase can catalyze the hydrolysis of the first PeOeP anhydride bond in ATP to produce intermediate ADP. The enzyme can also catalyze the hydrolysis of the PeOeP anhydride bond of ADP to finally produce AMP. AMP contains no phosphoanhydride bond, which may be the reason why AMP is not a substrate of AA-NADase. Fig. 10

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shows that AA-NADase also catalyzes the hydrolysis of the PeOeP phosphoanhydride bond in AMP-PNP to produce AMP, indicating that AA-NADase can hydrolyze the second PeOeP phosphoanhydride bond in ATP if the first one has been modified. Thus, AA-NADase is different from common ATPases. Although we cannot infer the detailed picture of the pathway of the AA-NADase-catalyzed hydrolysis of ATP, ADP and AMP-PNP from the present data, it is certain that AA-NADase could catalyze the hydrolysis of ATP, ADP and AMP-PNP to finally produce AMP. Fig. 11 shows that AMP specifically binds to AA-NADase. The binding of AMP to the enzyme may result from a combination of many specific interactions between the amino acid side chains of the enzyme and functional groups of the AMP molecule and results in the inhibition of the hydrolysis of NAD. 5. Conclusion The results presented above show that the highly purified AA-NADase has a unique multicatalytic hydrolysis activity, for it is not only able to cleave the CeN glycosyl bond of NAD to produce ADPR and nicotinamide, but also able to cleave the PeOeP bond of ATP, ADP and AMP-PNP to produce AMP. AA-NADase selectively cleaves the PeOeP bond of ATP, ADP and AMP-PNP without the cleavage of PeOeP bond of NAD. The hydrolysis reactions of NAD, ATP and ADP catalyzed by AA-NADase are mutually competitive. AA-NADase catalyzes the hydrolysis of ATP to produce AMP with an intermediate ADP. AA-NADase binds with one AMP with high affinity. AMP is an efficient inhibitor against NAD. AA-NADase has so far been identified as the first unique multicatalytic enzyme with both NADase and AT(D)Pase-like activities. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (Grant No. 20871111, 20571069, 20171041). We thank Dr. Qianrong Li for his assistance with the HPLC measurements. We also thank Professor Yangzhong Liu for advice on this paper. We thank two reviewers for their constructive comments, which helped improve the quality of this paper. References [1] P. Handler, J.R. Klein, The inactivation of pyridine nucleotides by animal tissues in vitro, Journal of Biological Chemistry 143 (1942) 49e57. [2] A. Masmoudi, P. Mandel, ADP-ribosyl transferase and NAD glycohydrolase activities in rat liver mitochondria, Biochemistry 26 (1987) 1965e1969. [3] H. Kim, E.L. Jacobson, M.K. Jacobson, NAD glycohydrolases: a possible function in calcium homeostasis, Molecular and Cellular Biochemistry 138 (1994) 237e243. [4] G. Orsomando, V. Polzonetti, P. Natalini, NAD(P)þ-glycohydrolase from human spleen: a multicatalytic enzyme, Comparative Biochemistry and Physiology 126 (2000) 89e98.

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