ATP Mimics pH-Dependent Dual Peroxidase-Catalase Activities Driving H 2 O 2 Decomposition

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Oct 2019ATP Mimics pH-Dependent Dual Peroxidase-Catalase Activities Driving H2O2 Decomposition Ying Shi, Menghuan Tang, Chaoqun Sun, Yadi Pan, Li Liu, Yijuan Long and Huzhi Zheng Ying Shi Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Beibei, Chongqing 400715 (China) Taiyuan University of Technology, Taiyuan 030024 (China) Google Scholar More articles by this author , Menghuan Tang Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Beibei, Chongqing 400715 (China) Google Scholar More articles by this author , Chaoqun Sun Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Beibei, Chongqing 400715 (China) Google Scholar More articles by this author , Yadi Pan Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Beibei, Chongqing 400715 (China) Google Scholar More articles by this author , Li Liu Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Beibei, Chongqing 400715 (China) College of Chemistry and Environmental Science, Qujing Normal University, Qujing 655011 (China) Google Scholar More articles by this author , Yijuan Long Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Beibei, Chongqing 400715 (China) Google Scholar More articles by this author and Huzhi Zheng *Corresponding author: E-mail Address: [email protected] Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Beibei, Chongqing 400715 (China) Google Scholar More articles by this author https://doi.org/10.31635/ccschem.019.20190017 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Adenosine triphosphate (ATP) is produced mainly in the mitochondrion, and its primary task is to function as a ubiquitous energy currency to meet the cellular metabolic demands in biological systems. Thus far, its potential role as performing enzymatic functions has not been elucidated. Herein, we investigated the pH-dependent dual-enzymatic properties of ATP, that is, its peroxidase- and catalase-mimetic activities in driving hydrogen peroxide (H2O2) decomposition in isolated mitochondria by employing Electron Spin Resonance spectrometry. In addition, we established a novel colorimetric assay for glucose sensing by combining conventional hexokinase action and the peroxidase-like activity of ATP. Our results showed that ATP imitated both intrinsic peroxidase and catalase activities through the decomposition of H2O2, accompanied by the formation of hydroxyl radicals under weak acid conditions and the generation of oxygen at neutral conditions, respectively. Quantum theoretical calculations indicated that the N7 atom of adenine of ATP serves as the binding site for the substrate, H2O2. Accumulating evidence revealed that H2O2 is one of the most important reactive oxygen species, and hence, its levels must be controlled to maintain redox homeostasis in cells. We found that mitochondrial membrane potential (MMP) of the isolated mitochondria, challenged with high concentrations of H2O2, followed by an addition of ATP, contributed significantly to H2O2 consumption to maintain the stability of MMP. Furthermore, the catalase-like activity of ATP markedly abrogated the cell damage triggered by ascorbic acid–induced oxidative stress. Thus, our findings demonstrate that the catalase-like activity of ATP in neutral condition and peroxidase-like activity in weak acid medium is presumably nature’s design to prevent MMP reduction elicited by a high concentration of H2O2 in the mitochondrial energy assembly in cellular systems. Download figure Download PowerPoint Introduction Adenosine triphosphate (ATP) is generally acknowledged as the “molecular unit of currency” for intracellular energy transfer, and it plays a fundamental role in many cellular metabolic pathways such as biomolecule synthesis and dissociation, muscle contraction, membrane transportation, and extracellular signaling.1–6 The process of ATP synthesis occurs mainly in the mitochondrion, and the abnormal concentration of ATP could, inevitably, lead to mitochondrial dysfunction and initiate many disease states. For example, lower production of ATP is an indicator of cellular vulnerability and injury, as its concentration declines very rapidly when cells undergo necrosis or apoptosis.7,8 Whereas an excessive production of ATP is related to cardiovascular diseases, malignant tumors, Parkinson’s and Alzheimer’s diseases.9–12 An accumulation of H2O2, a metabolic oxygen intermediate, is the most important representative of reactive oxygen species (ROS) in cells due to its long lifetime in diffusing to other cellular compartments.13,14 H2O2 is recognized as a messenger molecule, and its concentration level is related to the physiological balance of a living cell.15 Under a normal cellular redox cycle, excessive H2O2 is neutralized by H2O2-eliminating enzymes (catalase, glutathione peroxidase, nicotinamide adenine dinucleotide [NADH] peroxidase) and natural antioxidants (ascorbic acid [AA], glutathione).16,17 An imbalanced accumulation of H2O2 causes increased oxidative stress and thus induce cell damage, cancers development, as well as various neurological disorders such as Alzheimer‘s, Parkinson’s, and Huntington’s diseases.15,18–21 Thus, maintenance of the redox equilibrium to combat oxidative stress is vital for sustaining cellular homeostasis. Herein, we show that ATP has both pH-dependent peroxidase- and catalase-like activity, which enables H2O2 consumption. We showed that under weak acid conditions, ATP catalyzed H2O2 to oxidize substrate 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a color reaction. In neutral conditions, ATP functionally mimicked catalase to decompose H2O2 directly into H2O and O2. To the best of our knowledge, ATP is generated mainly in the mitochondria, as well as most of H2O2 during energy metabolism in most mammalian cells.22 Since the mitochondria are crucial intracellular regulators of energy metabolism, and the stability of the mitochondria is beneficial for the maintenance of normal physiological function of cells, we hypothesized that ATP might possess dual intrinsic catalytic activity, acting both as catalase and a peroxidase, to control H2O2 levels in the mitochondria. Thus, we sought to investigate the following: (1) Examine if ATP has a peroxidase-like activity in a catalytic oxidation reaction in the presence of H2O2 and in steady-state kinetics. (2) Investigate if ATP possess catalase-like activity using H2O2 as a source of dissolved oxygen and by Electron Spin Resonance (ESR) Spectroscopy to identify radical intermediates. (3) Determine plausible mitochondrial ATP-mediated intrinsic peroxidase-like activity, by employing a novel colorimetric platform for glucose sensing, constructed with the aid of hexokinase (HK). (4) Evaluate a plausible antioxidant enzyme-like activity of ATP by performing mitochondrial membrane potential (MMP) assays. (5) Examine if ATP could protect cells against endogenous H2O2-induced oxidative damage. Since the pioneering discovery of Fe3O4 magnetic nanoparticles with peroxidase-like activity in 2007 by Yan’s group,23 most nanomaterials have evoked increased interest to mimic peroxidase with the development of nanotechnology.24–30 Nanozymes as a promising candidate for artificial enzymes has attracted much attention due to their viable merits over natural enzymes, such as cost-effective synthesis, tunability in catalytic activities, and storage stability.29,30 Unfortunately, most of them are spontaneous aggregation in aqueous solutions, and the synthesis of nanomaterials often requires tedious operation steps.31 It has been reported that the catalytic activity of nanozymes is closely related to the components, particle size, morphology, and surface modification,30,32,33 which would cause variation of the catalytic activity from batch to batch and thus limit bulk production and application. ATP as a new type of mimetic enzyme is similar to nanozymes, with merits of low cost and good stability. However, ATP is commercially available to avoid tedious synthesis steps, and the catalytic activity difference in batches. Furthermore, the present study provides access to an innovative horizon of the physiological function of ATP and confirms that ATP has an attractive potential for other significant biomedical and biosensing applications of interest. Experimental Methods Enzyme mimicking activities of ATP The peroxidase-like activity of ATP was evaluated by the catalytic oxidation of organic substrate TMB in the presence of H2O2. Briefly, 100 μL of 100 μM ATP, 100 μL of 8.0 mM organic peroxidase substrate TMB, and 100 μL of 100 mM H2O2 were added into phosphate-buffered saline (PBS; pH 5.0), followed by 20 mM of TMB to a total volume of 1.0 mL. After incubation under the optimal conditions (40 °C for 30 min in the dark), with a subsequent color production, the absorption spectra were recorded at 652 nm on a UV-2450 spectrometer (Shimadzu, Japan), while monitoring the reaction kinetics at 1 min intervals and recording the absorbance over a period of 5 min. In our typical catalase-like activity assays, a reaction mixture containing 100 μL of varying concentrations of ATP (20 mM) diluted in 2.0 mL of PBS at pH 7.4, 100 mM H2O2 dissolved oxygen was used. Then the reaction was monitored, using a dissolved oxygen meter (DZS-708, SHJK JPSJ-606L) at 2 min intervals. ESR spectroscopy ESR spectroscopy was used to detect the generation of hydroxyl radical (·OH) with spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). All the ESR spectra were recorded on a Bruker ESR 300E, using the following settings: 20 mW microwave power, 1 G field modulation, and 100 G scan range. The assay of peroxidase-like activity of ATP was performed as follows: Samples containing 50 mM DMPO, 10 mM H2O2, at different concentrations of ATP were prepared in PBS at pH 5.0. Then the data were collected following 5 min incubation. The catalase-like activity of ATP was evaluated in the H2O2/UV experiment. Briefly, samples composed of 50 mM DMPO, 100 mM H2O2, and varying concentrations of ATP were prepared in PBS at pH 7.4. After exposure to UV light for 10 min, ESR spectra were immediately recorded. Computational estimation of ATP–substrate binding energies Quantum theoretical calculations were carried out to investigate the binding energy between the catalyst and the substrates. The geometries of catalyst, substrates, and their complexes were completely optimized via density functional theory (DFT) using the B3LPY/6-31g(d,p) basis sets,34–37 implemented in Gaussian 09 package.38 The binding energies (Eb) of substrate binding to catalyst through hydrogen bonding are defined as follows: E b = E Cat-Sub − ( E Cat + E Sub ) where ECat-Sub is the total energy of the complex formed through a hydrogen bond, ECat is the total energy of the catalyst, and ESub is the total energy of the substrate. ATP hydrolysis and free inorganic phosphate determination The extent of ATP hydrolysis was measured using a malachite green-ammonium molybdate assay.39 First, 500 μL of ATP (10 μM) was incubated at different temperatures for 40 min (test solutions), with a solution mixture without ATP kept at 4 °C to be used as a blank. Subsequently, malachite green stain cocktail, consisting of the stain (0.081% w/v), polyvinyl alcohol (2.3% w/v), ammonium heptamolybdate tetrahydrate (5.7% w/v in 6 M HCl), and water in a ratio of 2∶1∶1∶2, was dropped into 100 μL of the test and blank solution mixtures, followed by the addition of 40 μL of 3.4% sodium citrate to halt the hydrolysis of ATP. Finally, the mixture solutions were incubated at room temperature for 15 min, and the release of the inorganic free phosphate was recorded at an absorbance of 620 nm. Subsequently, a standard curve for phosphate was obtained using different concentrations of NaH2PO4 solutions, and the amount of inorganic phosphate generated by the ATP hydrolysis was calculated by extrapolation from the standard curve. HPLC analysis High-performance liquid chromatography (HPLC) was carried out using an Agilent 1290 system. The separation of components was performed on an Ultimate Plus-C18 column (5 μm, 4.6 × 150 mm) with a column oven temperature set at 37 °C. The mobile phases used were 90% buffer and 10% methanol. The flow rate was 1.0 mL·min−1. The separated substances were monitored and quantified at a wavelength of 254 nm. Cell incubation Breast cancer Michigan Cancer Foundation (MCF)-7 cells were purchased from ATCC (Maryland, USA) and were cultured in Dulbecco’s modified Eagle’s medium containing 100 U·mL−1 of penicillin and streptomycin. The medium and supplements were purchased from Gibco (California, USA). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. MMP assay Mitochondria were extracted from MCF-7 cells using a Cell Mitochondria Isolation Kit (Beyotime Co., Shanghai, China), as instructed by the manufacturer, described briefly as follows: (1) Cells were collected after trypsin digestion, and centrifuged at 150g for 7 min at 4 °C, dispersed in 2.0 mL of precooled PBS, and then centrifuged again at 600g for 5 min at 4 °C. (2) Precisely 1.5 mL of mitochondria separation reagent was mixed with ∼10 million cells, then cells were gently suspended in extraction buffer on an ice bath for 12 min. (3) The cell suspension was transferred to a glass homogenizer (Shanghai LABSEE Biological Co., Ltd., Shanghai, China) and homogenized 10 times by piston grinding. The cell homogenate was centrifuged at 600g for 10 min at 4 °C. (4) The supernatant was carefully transferred to 10 mL centrifuge tube and centrifuged at 8000 revolutions per minute (rpm) for 4 min to acquire sediment-isolated mitochondria, and then resuspended in PBS. Subsequently, MMP was estimated using JC-1 (Beyotime Co., Shanghai, China), a cationic dye probe that acts by penetrating energized mitochondrial membranes, while accumulating gradually. Thus JC-1 sensitivity to MMP reflects a healthy status of the mitochondria. At high membrane potentials, JC-1 forms aggregates, with a resultant red-fluorescence in the mitochondrial matrix; conversely, at lower potentials, the dye exists as a monomer, yielding green fluorescence.40 Briefly, after treated with different concentration of ATP and H2O2, mitochondria were incubated at 37 °C for 30 min with 5 mg·L−1 JC-1. Finally, MMPs were monitored by determining dual emissions from mitochondrial JC-1 monomers (emission at 530 nm under 490 nm excitation) and aggregates (emission at 590 nm under excitation of 540 nm). The ratios of red to green fluorescent intensities were calculated to evaluate the mitochondrial activity. Cell viability A cell count kit-8 (CCK-8; Beyotime Co., Shanghai, China) was employed to evaluate the cell viability. First, MCF-7 cells were seeded into 96-well plates (5000 cells per well) for 24 h incubation. After that, ATP and AA, at the indicated concentrations, were added to the cell culture medium and incubated with cells for 6 h. Finally, cells were washed with PBS, and CCK-8 was added for further 60 min incubation at 37 °C. The absorbance at 450 nm was determined using an Infinite 200 pro microplate reader (Tecan, Austria). Glucose sensing by coupling ATP and HK reactions Glucose detection was performed by a facile two-step method. First, 10 μL of 500 μM ATP, 10 μL of 100 U·L−1 HK, 10 μL of 50 mM MgCl2, and tubes containing 10 μL of glucose at different concentrations, ranging from 0 500 μM, was mixed with 10 μL of 50 mM pH 7.4 Tris–HCl buffer for an incubation time of 60 min at 37 °C. After that, 100 μL of 8.0 mM TMB, 100 μL of 100 mM H2O2, 100 μL of pH 5.0 PBS, and water was added to each tube to a volume of 1.0 mL. Finally, the mixture of each tube was incubated at 50 °C for 40 min, and the catalytic oxidation of the peroxidase substrate TMB (oxTMB) of each sample was measured at an absorbance of 652 nm, and utilized as an index to assess the peroxidase-like activity of ATP. Based on the above coupling reaction process, human blood serum glucose levels were effectively monitored as follows: The normal human blood samples were taken from two healthy volunteers. Informed consent was sought from each of the volunteers and a research approval was obtained from the Institutional Research Ethics Committee of Southwest University hospital. The blood samples were centrifuged at 3000 rpm for 30 min, serum was collected from each sample and then treated by ultrafiltration using a 10 kDa ultrafiltration membrane and then centrifuged at 3500 rpm for 20 min to remove light-absorbing plasma factors that might interfere with the assay. The filtrate from each sample was collected, diluted 10 times, and the TMB oxidation measurement was repeated at an absorbance of 652 nm. Results and Discussion Dual-enzyme mimicking activities of ATP The peroxidase-like activity of ATP was estimated with a chromogenic substrate TMB. Figure 1a shows that ATP catalyzed H2O2-mediated oxidation of TMB at pH 5.0, along with a characteristic absorption peak at 652 nm and the occurrence of blue color of the reaction mixture (Figure 1b). For the control experiments, set in separate tubes without one of the reaction components, ATP or TMB or H2O2, color variation and absorbance change were negligible, which indicated that the peroxidase-like activity of ATP under weakly acidic conditions took place under the test experimental conditions. The initial rate of the oxidization of TMB had a linear relationship with increased concentration of ATP in the range from 2.0 to 6.0 μM; then the increment of the reaction rate gradually decreased as ATP concentration continued to increase (), imitating first-order kinetics. The peroxidase-like activity of ATP is pH-dependent, with ATP showing peroxidase-like activity in weak acidity condition and indeed deactivated when the pH inclined to neutral condition (~pH = 7.4) (). Besides, the peroxidase-like activity of ATP evaluated at varying pH from 1 to 7 or H2O2 concentration from 0 to 50 mM or temperature from 10 to 80 °C (), demonstrated that the peroxidase-like activity of ATP was also dependent on H2O2 concentration and temperature, which correlated with the properties of the natural HRP enzyme and other reported peroxidase-like nanomaterials.23,41,42 Figure 1 | The peroxidase-like activity of ATP. The UV-visible absorption spectra (a) and the corresponding photographs (b) of ATP catalyzed the oxidation of 3,3′,5,5′-TMB in the presence of H2O2 at pH 5.0. Download figure Download PowerPoint To further investigate the peroxidase-like activity of ATP, its steady-state kinetic behavior was determined, and the resulting curves, indeed, followed the Michaelis–Menten kinetics (). The Michaelis–Menten constant (Km) and maximum velocity (Vmax) were obtained from the Lineweaver–Burk plot (). The Km value is an indicator of enzyme affinity to substrates, and a lower Km value implies a stronger affinity. Vmax reflects the maximal reaction velocity. The catalytic constant kcat is the ratio of the maximum velocity and enzyme concentration, and it is a standard typically used to assess the velocity of an enzymatic-catalyzed reaction at a fixed enzyme concentration. Compared with other reported small molecular peroxidase mimetics, such as guanosine triphosphate (GTP),43 fluorescein,31 and its derivatives [5(6)-carboxyfluorescein, 5(6)-aminofluorescein, 2’,7’-difluorofluorescein, 2′,7′-dichlorofluorescein],44–46 as well as acidic amino acids including L-glutamic acid and L-aspartic acid,47 ATP had a high binding affinity toward H2O2, except for 2′,7′-dichlorofluorescein and acidic amino acid. Moreover, the Km value of ATP was lower than that of HRP when compared, using H2O2 as the substrate, which indicated that ATP has a better affinity to H2O2 than the natural enzyme. The catalytic constant kcat of ATP was two orders of magnitude higher than that of fluorescein and its derivatives, and four orders of magnitude higher than that of acidic amino acids, supporting the high catalytic activity of ATP, when compared with other reported small molecular catalyst enzyme mimics such as DNA-conjugated small enzyme and l-Proline enzyme mimics. However, the kcat value of ATP was lower than that of GTP and HRP, implying a relative weaker catalytic activity of ATP relative to these two biological molecules. In addition to the peroxidase activity, ATP also exhibited catalase-like activity by decomposing H2O2 directly into H2O and O2 under neutral condition. A dissolved oxygen meter was employed to measure the oxygen generation from the H2O2 decomposition in the presence of ATP. H2O2, a binary weak acid, gradually ionized in water, as follows: H 2 O 2 ⇄ H + + HO 2 − HO 2 − ⇄ H + + HO 2 2 − In an alkaline environment, hydrogen ion (H+) is neutralized by hydroxyl ion (OH), resulting in ionization equilibrium shifting to the right, which is favorable for producing peroxide ion (O22−), more likely to decompose to generate oxygen than to form H2O2.48 As displayed in Figure 2a, at pH 7.4, dissolved oxygen in H2O2 solution showed only a slight increase in dissociation with time, whereas the concentration of dissolved oxygen in an ATP–H2O2 reaction solution demonstrated a higher dissociation, compared with the solution without ATP, and significantly increased with gradual enhancement of ATP under the relevant physiological conditions. In contrast, under acidic conditions, the ionization equilibrium was inhibited and led to reverse migration, with the equilibrium shifted toward H2O2 formation. As shown in Figure 2b, at pH 5.0, the equilibrium toward H2O2 dissolved oxygen generation dropped slightly initially and then reached a saturation value as ionization balance was achieved. Notably, dissolved oxygen generated in ATP–H2O2 reaction systems decreased markedly with time and with increased ATP concentration, which attributed to the peroxidase-like activity of ATP under acidic condition, and thus, led to H2O2 consumption and the disturbance in the kinetic equilibria. These results demonstrated that ATP could also serve as catalase under physiologically relevant conditions. The kinetic analysis of the catalase-like activity of ATP was also investigated, which fitted the classical Michaelis–Menten model (). Subsequently, the kinetic parameters (Km and kcat value) of the ATP-catalase mimetic were obtained from the Lineweaver–Burk plot () and compared with those of the natural enzyme, catalase. As listed in , both the Km value of ATP-catalase-like activity was lower than that of the natural catalase enzyme, suggesting that H2O2 substrate affinity for ATP is better than the natural catalase enzyme, whereas substrate decomposition turnover number is weaker for ATP than catalase. Figure 2 | The catalase-like activity of ATP. Time-course curves of dissolved oxygen generation from H2O2 catalyzed at different ATP concentrations in pH 7.4 (a) and pH 5.0 (b) PBS buffer. Concentrations of H2O2 were all 100 mM, and the concentrations of ATP were 0 mM (⊳), 0.10 mM (⋄), and 0.20 mM (⋆), respectively. Download figure Download PowerPoint Study of the catalytic mechanism of ATP To understand the intriguing dual-enzymatic activities of ATP, we investigated the possible catalytic mechanisms of reactions using the ESR technique. The spin trap DMPO could capture the short-lived, highly reactive ·OH radical to form DMPO/·OH spin adduct with a characteristic spectrum of 1∶2∶2∶1 intensity.49 As shown in Figure 3a, no DMPO/·OH spin adduct was observed in the presence of H2O2 at pH 5.0. On the other hand, signal intensity was produced with added ATP and intensified with a gradual increase in ATP concentration, indicating that ATP is competent in catalyzing H2O2 to generate ·OH under weakly acidic conditions. The ESR spectra of H2O2 in the presence of DMPO under UV illumination were employed to investigate the catalase-like mechanism of ATP at pH 7.4. Since it is a well-known fact that ·OH is produced when H2O2 is irradiated with UV light, we anticipated that the H2O2 decomposition catalyzed by catalase should reduce the UV-induced ·OH production from H2O2. Therefore, ESR signals could be applied to verify the catalase-like activity of ATP. As shown in Figure 3b, a prominent signal was produced upon UV-light illumination in the presence of H2O2, whereas the ESR signal induced by ·OH was gradually reduced in an ATP concentration-dependent manner, suggesting the depletion of H2O2 by ATP, and thus, confirmed its ATP-catalase-mimic activity. Figure 3 | Effect of ATP on ·OH production using the substrate H2O2 in a concentration-dependent manner. (a) H2O2 (10 mM)/DMPO system in pH 5.0 PBS after 5 min incubation; (b) UV/H2O2 (100 mM)/DMPO system in PBS at pH 7.4 and exposure to UV light for 10 min. Download figure Download PowerPoint Numerous literature reports have indicated that the addition of ATP and adenine could improve the catalytic activity of DNAzyme and nanozyme.39,48,50 These studies demonstrated that adenine with a structure similar to the indole group of the histidine residue in the natural peroxidase enzyme might play a histidine-like H2O2-activator role in protein peroxidases, which leads to their enhanced proficiency in catalysis. We speculated that adenine molecules might act as binding sites for the substrate H2O2 in our studies. Thus a quantum theoretical calculation was applied to verify the catalytic mechanism. As shown in , the highest occupied molecular orbital for ATP is occupied mainly by the base, adenine, indicating that adenine might act as proton receptor to promote the binding of the enzyme to the substrate H2O2, consistent with the observation made with the role played by distal histidine of the native enzyme. For a better understanding, the role of the adenine group in catalysis and the binding modes between adenine and H2O2 molecule were investigated. As shown in Figure 4, the binding energy (Eb) of N7 atom of adenine upon H2O2 exposure was lower than those of other N-binding modes in the molecule, implying that the N7 atom of adenine likely serves as the binding site for H2O2. Altogether, our findings led us to propose possible reaction modes of ATP mimicking the roles of catalase and a peroxidase (), Scheme 1. Figure 4 | Optimized structures of hydrogen bonding between different sites of the adenine base of ATP and the substrate H2O2 obtained from computed binding energy (Eb) studies. (a) N7 atom of adenine with H2O2, (b) N7 atom and amino group of adenine with H2O2, (c) N2 atom and the amino group of adenine with H2O2, and (d) N2 atom of adenine with H2O2. represents H atom, represents C atom, represents N atom, represents O atom, and represents P atom. Download figure Download PowerPoint Scheme 1 | Illustration of ATP with pH-dependent dual-enzymatic activities driving H2O2 decomposition. (a) The catalase-like activity allows ATP to profoundly rescue the mitochondria and whole cell from oxidative stress damage. (b) A novel colorimetric assay is established for glucose sensing by combining hexokinase and the peroxidase-like activity of ATP. Download figure Download PowerPoint Catalytic activity is due to intact ATP It has been reported that the energies released from ATP hydrolysis could accelerate a catalytic reaction.39,51 In order to exclude that the hydrolysis of ATP rendered its ability to promote the H2O2 decomposition, we evaluated the degree of ATP hydrolysis in our model system by measurin