Inhibition of Catalase by Tea Catechins in Free and Cellular State: A Biophysical Approach

Tea flavonoids bind to variety of enzymes and inhibit their activities. In the present study, binding and inhibition of catalase activity by catechins with respect to their structure-affinity relationship has been elucidated. Fluorimetrically determined binding constants for (−)-epigallocatechin gallate (EGCG) and (−)-epicatechin gallate (ECG) with catalase were observed to be 2.27×106 M−1 and 1.66×106 M−1, respectively. Thermodynamic parameters evidence exothermic and spontaneous interaction between catechins and catalase. Major forces of interaction are suggested to be through hydrogen bonding along with electrostatic contributions and conformational changes. Distinct loss of α-helical structure of catalase by interaction with EGCG was captured in circular dichroism (CD) spectra. Gallated catechins demonstrated higher binding constants and inhibition efficacy than non-gallated catechins. EGCG exhibited maximum inhibition of pure catalase. It also inhibited cellular catalase in K562 cancer cells with significant increase in cellular ROS and suppression of cell viability (IC50 54.5 µM). These results decipher the molecular mechanism by which tea catechins interact with catalase and highlight the potential of gallated catechin like EGCG as an anticancer drug. EGCG may have other non-specific targets in the cell, but its anticancer property is mainly defined by ROS accumulation due to catalase inhibition.

The ability of flavonoids to bind and inhibit some vital cellular enzymes leading to suppression of cell proliferation is being investigated widely [18][19][20][21][22]. EGCG is known to bind to proteins like salivary proline-rich proteins, fibronectin, fibrinogen and histidine rich glycoproteins [23]. Caseins and lactoglobulins are milk proteins which rendered the antioxidant activity of tea polyphenols upon binding with catechins [24][25][26][27]. EGCG binds to Bcl-2 proteins with inhibition constant (K i ) 0.33-0.49 mM and to vimentin and G3BP1 with dissociation constant (K d ) 3.3 nM and 0.4 mM, respectively [28][29][30]. In the present study, binding of catechins to catalase -an enzyme which maintains the cellular ROS levels is followed fluorimetrically and thermodynamically characterized.
Catalase is an antioxidant oligomeric enzyme (MW 2,40,000) with four identical subunits arranged tetrahedrally [31][32][33]. Each subunit consists of a single polypeptide chain that associates with a prosthetic group, ferric protoporphyrin IX [34]. It is a very important enzyme in protecting cells from oxidative damage by ROS. Drugs bind to enzymes and elicit enzyme inhibition; one such example is catalase inhibition by wogonin led to H 2 O 2 accumulation and cytotoxicity in cancer cells through H 2 O 2mediated NF-kB suppression and apoptosis activation [35]. Similarly, EGCG at concentrations between 5-20 mM also inhibited phosphorylation of JNK, JUN, MEK1, MEK2 in JB6 epidermal cell lines [36]. In MCF7 breast cancer cell lines, cyclin dependent kinase 2 (CDK2) and CDK4 were reported to be inhibited by 30 mM EGCG [37]. In KYSE 510 human esophageal cancer cells, EGCG inhibited DNA methyltransferase with K i 7 mM [38]. All these enzymes have relevance in cancer prevention. Inhibition of human cancer cell growth by tea polyphenols has been observed in H1299, H661, HT-29, H441 and breast cancer cell lines at IC 50 values between (20-75 mg/ml) [39]. Catalase is another cellular enzyme whose interaction with microsystin, a cyanotoxin drug, decreases its enzymatic action [40]. Here its inhibition in K562 cancer cells is reported and its relevance in cancer is demonstrated by the suppression of cell viability. The study also highlights the influence of structure of the catechins on the inhibition of catalase activity by catechins.
As endogenously formed ROS are important in promoting carcinogenesis, tea polyphenols may have important role in quenching them and also tea polyphenols being redox active can under go auto-oxidation and produce ROS in media and in mitochondria [41,42]. This makes activity of tea polyphenols in cancer prevention very complex which needs active investigation. Changes in ROS population are expected manifestation of catalase inhibition and have been followed to monitor the enzyme activity on EGCG treatment.

Cell culture
K562 (human chronic myeloid leukemia cell line) was procured from NCCS, Pune and grown in RPMI 1640 supplemented with 10% FBS, 100 U/ml of penicillin and 100 mg/ml of streptomycin in 25 cm 2 T-flasks (Nunc, Roskilde, Denmark) with vented caps and incubated at 37uC in a humidified atmosphere of 5% CO 2 in air.

Fluorescence spectroscopy
Fluorescence spectra were recorded using a Perkin Elmer fluorescence spectrophotometer (LS-55) equipped with 150 W Xenon flash lamp and using fluorescence-free quartz cell of 1 cm path length. The widths of excitation and emission slits were set to 5 nm, and scan speed (100 nm/min), excitation voltage were kept constant for each data set. Quantitative analysis of the potential interaction between catechins and catalase was performed by the fluorimetric titration technique. Briefly, solution of catalase (5610 27 M, as calculated using molar extinction coefficient of 3.24610 5 M 21 s 21 at 405 nm for catalase) in 50 mM phosphate buffer of pH 7.4 was titrated in cuvette by successive additions of individual catechin solution aliquots from a stock of 5610 27 M. Fluorescence emission spectra were recorded in the wavelength range of 290-450 nm upon excitation at 280 nm when catalase samples were titrated with catechins. All experiments were carried out at room temperature. Fluorescence intensity was measured at 340 nm of protein emission spectra. Fluorescence spectra of individual catechins in buffer were recorded as blanks under the same experimental conditions and subtracted from the corresponding sample to correct the fluorescence background.

Isothermal titration calorimetry (ITC)
The energetics of the binding of catechins to catalase was studied by ITC using a MicroCal ITC 200 , (Northampton, MA, USA). All solutions were degassed under vacuum (140 mbar, 10 min) on the MicroCal's Thermovac unit to eliminate air bubble formation inside the calorimeter cell. Briefly, the calorimeter syringe was filled with a concentrated solution of catechins (10 mM each). Successive injections of this solution into 1 mM solution of catalase in the calorimeter cell were effected from the rotating syringe with constant stirring of the solution. The titration and analysis were performed through Origin 7 software provided with the unit.

Circular dichroism (CD) spectroscopy
All the CD experiments were carried out on a Jasco-815 automatic spectropolarimeter (Jasco International Co., Ltd. Hachioji, Japan) equipped with a peltier cuvette holder and temperature controller PFD425 L/15. The catalase concentration and path length of the cuvette used were 1 mM and 0.1 cm, respectively. The instrument parameters were set at scanning speed of 50 nm/min, bandwidth of 1.0 nm and sensitivity of 100 milli degree. The molar ellipticity values are expressed in terms of mean residue molar ellipticity, in units of deg. cm 2 dmol 21 .

Inhibition of catalase activity
Absorption spectrophotometer (Varian, CARY 100 Bio, USA) was used to determine pure catalase activity (cell free). Briefly, 900 ml of H 2 O 2 (0.036%) was taken in quartz cell of 1 cm path length and its absorbance was recorded at 240 nm. To the solution, 100 ml of catalase (50 U/ml) was added and the decrease in absorbance was recorded at 10 s interval. This experiment was performed in presence 50 mM each of four catechins and 3-AT separately to evaluate the inhibitory effect of catechins on catalase activity, after 1 h incubation.
Estimation of cellular catalase activity was carried out by suspending K562 cells (5610 4 for each sample) in 1 ml of PBS along with four catechins and 3-AT (50 mM each) separately and incubated at 37uC for 2 hours. Later, each sample was centrifuged and washed twice with PBS. The final pellet was suspended in 200 ml lysis buffer and kept on ice for 30 min. The lysate was then used in the same protocol used for evaluation of pure catalase activity.

Analysis of cell viability
Cell viability of K562 cells was quantified by MTT based colorimetric assay as described elsewhere [43]. Briefly, cells were rinsed with PBS and 0.5 mg/ml MTT was added into each sample. The mixture was incubated at 37uC for an additional 3 h. At the end of incubation period, the medium containing MTT was removed and the pellet was dissolved in 200 ml DMSO. Absorbance was measured at a wavelength of 570 nm. Cell viability was expressed as a percentage of the control culture.

Measurement of intracellular ROS
Intracellular ROS were estimated by using the DCFDA fluorescent probe [9]. Intracellular H 2 O 2 and other peroxides can oxidize DCFDA to highly fluorescent compound dichlorofluorescein (DCF). K562 cells were incubated at 37uC in absence and presence of EGCG (12.5 -100 mM) for 24 h. Cells were again incubated with 10 mM DCFDA at 37uC for an additional 30 min, and then washed twice with PBS. Finally the fluorescence intensity of DCF was measured with an excitation wavelength of 485 nm and emission wavelength of 530 nm.

Statistical analysis
Data were analyzed by Origin Software (Version 8) for Windows. The statistical analysis of the samples was undertaken using Student's t-test. All data reported are means 6 standard deviations for three independent experiments, unless otherwise noted.

Quenching of catalase fluorescence by catechins
Catalase shows a fluorescence emission peak at 340 nm with the excitation wavelength at 280 nm, mainly due to the presence of tryptophan and tyrosine residues [44]. This emission peak exhibited progressive decrease in intensity on addition of catechins suggesting interaction between catalase and catechins (  ITC directly measures the heat released during a chemical reaction. ITC can reveal the stoichiometry, enthalpy free energy, and entropy changes that occur over the course of a reaction [45,46]. Data obtained from the titration are presented by the series of peaks corresponding to each injection. Usually, this representation is transformed into the apparent heat change between two injections as a function of the titrant ratio by means of integration of power differential with regard to time. ITC has been used mainly to quantify interactions in various biochemical systems [47][48][49]. ITC has been used in the present study to determine binding affinity and other thermodynamic parameters of catechins-catalase interaction. Representative isothermal calorimetric heat profiles for the binding of catechins-catalase interaction, at physiological pH and 25uC are shown in Figure 4 (A-D). The data were corrected for the heat of dilution of catechins, which was determined in a separate set of experiments under identical conditions. The titration and analysis were performed using one-site binding model through Origin 7 software provided with the unit. The thermodynamic parameters recorded for the binding of catechins (10 mM each) with catalase (1 mM) are summarized in Table 1. From Figure 4 it is observed that the titration of catalase with all the four catechins yield negative heat deflections. EC, EGC, ECG, and EGCG bind to catalase with binding constants 2.13610 5 , 1.92610 5 , 6.36610 5 , and 8.19610 5 M 21 , respectively, as determined by the following equation: DG = 2RT ln K, where DG is the free energy, R and T are the gas constant and temperature, respectively, K is the binding constant.

Catechin induced conformational changes in catalase
CD allows investigation of the conformational changes that occur in a protein upon ligand binding [50]. The CD spectra of catalase at pH 7.4 in the absence and in the presence of EGC, EC, ECG, EGCG, and 3-AT at 25uC are represented in Figure 5 (af). The CD spectra of catalase exhibited two negative bands in the far-UV region at 208 and 220 nm, characteristic of an a-helical structure of protein [51]. The negative band at 208 and 220 nm decreased in intensity with the addition of various catechins, which is indicative of the loss of a-helicity upon interaction. The CD spectra of catalase in presence and absence of catechins were observed to be similar in shape, indicating that the structure of catalase is predominantly a-helical [52]. From the CD spectra, the effect of catechins on the secondary structure of catalase is in the following order: 3-AT.EGCG.ECG.EC.EGC.

Inhibition of catalase activity
The spectroscopic approach for determination of catalase activity on H 2 O 2 degradation showed continuous breakdown of its substrate, H 2 O 2 with time (upto 100 s) at 240 nm. On addition of catechins, a decrease in rate of H 2 O 2 degradation i.e. a decrease in pure catalase activity was observed which is represented in Figure 6A. Catechins with galloyl moiety (ECG and EGCG) reduced catalase activity more than its non-gallated counterparts i.e. EGC and EC. However, Inhibition by EGCG is the closest to inhibition by known catalase inhibitor 3-AT. The order of inhibition of catalase activity was found to be 3-AT.EGCG. ECG.EC.EGC. The same experiment with cellular catalase in K562 cells revealed inhibition of cellular catalase activity in the presence of catechins ( Figure 6B). The trend of inhibition in this condition was observed to be similar to that of pure catalase. The double reciprocal plot or Lineweaver-Burk plot shows that the inhibition of catalase by EGCG is of uncompetitive type (Figure 7). The y-intercept i.e. 1/V 0 value for uninhibited and inhibited catalase were found to be 0.0081 and 0.3635 mM 21 min, respectively.

MTT assay for cell viability
The concentration (IC 50 ) at which the K562 cell survivability was reduced to 50% was calculated for EGCG and 3-AT by MTT based cell viability assay. The IC 50 value obtained for EGCG and 3-AT was 54.5 mM and 10.5 mM, respectively.  (Figure 8).

Discussion
Catechins are antioxidants ubiquitously found in green tea and among them EGCG shows highest antioxidant activity that may have therapeutic applications in cancer treatment. EGCG functions as a powerful antioxidant, preventing oxidative damage in healthy cells, but also as an anti-angiogenic and antitumor agent and as a modulator of tumor cell response to chemotherapy. However, neither pro-oxidant nor antioxidant activities have yet been clearly established to occur in vivo in humans [53]. Natural polyphenols are reported to be good inhibitors of human dihydrofolate reductase (DHFR) could explain the epidemiological data on their prophylactic effects for certain forms of cancer and open a possibility for the use of natural and synthetic polyphenols in cancer chemotherapy [54].
Fluorescence quenching experiments demonstrated that the ester bond containing tea polyphenols EGCG and ECG are effective inhibitors of DHFR with K d of 0.9 and 1.8 mM, respectively, while polyphenols lacking the ester bond containing galloyl moiety (e.g., EGC and EC) did not bind to this enzyme [54]. The binding constant of EGCG to catalase was determined fluorimetrically to be 2.27610 6 M 21 (K d of 0.44 mM). Other catechins were not as effective as EGCG establishing the influence of galloyl moiety for efficient binding. The binding efficiency is attributed to the eight phenolic groups of EGCG which serve as hydrogen bond donors. Involvement of positive charge on the carbon atom of the ester bond with the electronegative groups of amino acid residues like histidine also contributes towards efficient binding. Here it can be reasoned that at this concentration of EGCG it can also bind to other non-specific targets in vivo due to the presence of OH groups. To this end, it can be emphasized that binding to catalase inhibits its activity and increases the ROS population which eventually triggers apoptosis. It is known that 3-AT (an efficient inhibitor of catalase) binds with histidine residue (His75) near heme group of catalase forming a non-coplanar adduct (very close to Tyr358). Loss in fluorescence intensity of catalase suggests involvement of EGCG cation with histidine anion (pKa = 6.5) [55] near tyrosine residue (Tyr358) of active site [56]. This identification of the important structural features responsible for inhibition will provide valuable information for designing new inhibitors.
ITC provides valuable information on biomacromoleculeligand interactions [57,58]. This technique measures the heat released or absorbed (DH) when the macromolecule binds to  [40], which is lower than catalase-EGCG complex. Microcystin binding to catalase was reported to influence its physiological functions and conformation [40]. Binding is stronger with EGCG and more changes are observed in catalase-catechins complexes. Loss of a-helix is identified in the CD spectra by the decrease in negative peak at 222 nm and 208 nm (signature peaks of a-helix) on addition of EGCG as shown in Figure 5. Gain in bsheet is observed from the increase in negative peak at 218 nm against 222 nm for a-helix. These conformational changes in catalase secondary structure on interaction with catechins influence its active site and physiological functions, rendering it inactive in scavenging ROS.
In the present study, the physiological changes are demonstrated by the inhibition of catalase activity by catechins. EGCG is observed to inhibit more efficiently than ECG and other nongallated catechins. From the decay curves as shown in Figure 6, it is inferred that the rate of H 2 O 2 degradation is reduced by catechins in cell free ( Figure 6A) and cellular state ( Figure 6B). From the Lineweaver-Burk plot (Figure 7), it is evident that the maximum inhibition of catalase is achieved by EGCG. The plot also demonstrates uncompetitive type of inhibition suggesting that EGCG binds to catalase at a site other than the active site. As a consequence, the uncompetitive inhibitor (here EGCG) lowers the measured maximum velocity (V max ) and also decreases apparent K m value [59]. This inhibition in catalase activity results in   accumulation of ROS as illustrated in Figure 8. Higher levels of ROS create oxidative stress which is detrimental to cellular integrity.
The IC 50 value for suppression of cell viability in K562 cells on EGCG treatment is determined to be 54.5 mM (or 27 mg/ml). Inhibition of human cancer cell growth by tea polyphenols has been observed in H1299, H661, HT-29, H441 and breast cancer cell lines at IC 50 values between 20-75 mg/ml [39]. Higher EGCG concentration is required in the cell to elicit cellular response than its K d value to catalase. Here it can be emphasized that suppression of cancer cell viability is not envisaged only by catalase inhibition but other pathways contribute to it. EGCG is capable of generating H 2 O 2 in solution by auto-oxidation which contributes to oxidative stress. Superoxide is also reported to be generated in EGCG containing solutions [60]. In such conditions, inhibition of catalase by EGCG further increase the oxidative stress triggering apoptosis and eventually leading to cell death. These phenomena can be therapeutically used for cancer prevention. These revelations suggest that a balance between all the major activities of EGCG like antioxidants, pro-oxidants and enzyme inhibitors leads to cellular suppression, which is again cell specific. Inhibition of cellular enzymes by catechins has found major pharmacological applications as anticancer drugs [61]. Addition of methyl groups to ECG enhanced binding to DHFR and is a new approach to find novel inhibitors from natural resources [54]. A concentration of 27 mg/ml is high for EGCG to be bioavailable by tea consumption. Here EGCG is then treated as drug that can be delivered in various forms; one of which could be nano-formulations.

Conclusion
The efficacy of EGCG to bind with catalase and render its activity in cancer cells definitely represents a fascinating tool in the field of oncology. The present work sheds light on the critical structural features of galloylated catechins and identifies probable mechanism by which they inhibit catalase. These findings provide guidance for designing efficient catalase inhibitors. However, a cancer cell death event is an endpoint of several pathways and further studies in this context are in progress to completely understand the role of catechins in anticancer therapy. This study also demonstrates that natural polyphenols could be used as 'guide compounds' for development of new anticancer drugs.