A study of the inhibition of catalase by dipotassium trioxohydroxytetrafluorotriborate K₂[B₃O₃F₄OH]

Abstract

In the development of boronic acid-based enzyme inhibitors as potential pharmaceutical drugs, dipotassium trioxohydroxytetrafluorotriborate K₂[B₃O₃F₄OH] was listed as a promising new therapeutic for treatment of these diseases. The catalase-mediated conversion of hydrogen peroxide, in the presence and absence of K₂[B₃O₃F₄OH] was studied. The kinetics conformed to the Michaelis–Menten model. Lineweaver–Burk plots were linear and plotted the family of straight lines intersected on the abscissa indicating non-competitive inhibition of the catalase. It appears that in the absence of inhibitor, catalase operates the best at conditions around pH 7.1 and in the presence of K₂[B₃O₃F₄OH] the optimum is around pH 6.2. The uncatalyzed reaction of hydrogen peroxide decomposition generally has a value of activation energy of 75 kJ mole⁻¹, whereas catalase, in the absence of inhibitor, lowers the value to 11.2 kJ mole⁻¹, while in the presence 69 mmoles L⁻¹ of K₂[B₃O₃F₄OH] it was 37.8 kJ mole⁻¹.

• Boroxine
• catalase
• drug research
• enzyme inhibition

Introduction

Halogenated boroxines belong to derivatives of cyclic anhydride of boronic acid1. It has been suggested that halogenated boroxines can be used in the prevention and/or treatment of benign or malignant changes of the epidermis visible in the form of, for example, nevus or skin cancer. In previous works2–4, it was shown the effects of dipotassium trioxohydroxytetrafluorotriborate (K₂[B₃O₃F₄OH]) on genetic material and inhibition of cell division in human cell cultures. Tested concentrations (0.04, 0.1, 0.2 and 0.4 mg mL⁻¹) were correlated with inhibition of cell growth in basal cell carcinoma culture and with the lymphocytes proliferation. Clastogenic activity has been confirmed, without evidences of aneugenic activity in human lymphocytes. As there are not enough data in the recent literature about the bioactivity of this compound we continued our investigation. Our preliminary results of in vitro and in vivo antitumor investigation of K₂[B₃O₃F₄OH] showed very expressive antitumor activity (yet not published) that is comparable to the well-known antitumor drug 5-fluorouracil.

Due to their unique electronic structures, boronic acid compounds can be used for the development of enzyme inhibitors and consequently various compounds of this kind have been widely studied in this role. This intensive area of medicinal chemistry research has recently culminated in the commercialization of the peptidyl boronic acid antineoplastic drug Velcade5,6. In the development of boronic acid-based enzyme inhibitors as potential pharmaceutical drugs, target specificity within a wide family is crucial to avoid side effects. Following these investigations and statements, we decided to investigate the inhibitory properties on catalase activity of K₂[B₃O₃F₄OH], a compound promising as a new therapeutic for treatment of cancer and inflammatory diseases.

It is well-known that intracellular antioxidant mechanisms involve antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase in tissues. Hydrogen peroxide (H₂O₂) is a very reactive oxygen species and plays a role in the pathologies of many diseases. The catalase is an antioxidant enzyme found in many cell types and considered to play a major role in the removal of H₂O₂7,8. This enzyme speeds up a reaction which breaks down toxic H₂O₂ and can protect cell membranes and DNA from this damaging oxidation process. This reaction is important to cells because H₂O₂ is produced as a byproduct of many normal cellular reactions and loss of catalase activity is associated with increased susceptibility to oxidative stress. The strong links between oxidative stress and different disease disorders plus the protective role of catalase in these disorders suggests that any endogenous inhibitor of catalase has potential to contribute detrimentally to the disease pathology. Recently, it has been shown that faulty cellular antioxidant systems cause organisms to develop a series of inflammatory and cancer diseases. Reduced catalase activity is a feature of Alzheimer’s9, diabetes10, atherosclerosis11 and cancer diseases12 raising the possibility of a direct link between elevated concentration of some compounds in the cell and reduced catalase activity13.

Although catalases have been studied for years, there are many interesting questions still unsolved regarding their function in presence of different inhibitors as what is the kinetic mechanism of inhibition and what are the kinetic parameters? Since the presence of potential drugs in the cell can reduce the activity of catalase, in our study, we wanted to investigate the H₂O₂ kinetic parameters and inhibition mechanism of catalase in presence of K₂[B₃O₃F₄OH] with the aim to show how this substance affects catalase activity.

Materials and methods

Materials

The catalase from bovine liver was purchased from Sigma-Aldrich (C100: 50 mg, 47 031 U mg⁻¹ protein). Stock solution is prepared by the dissolution 2 mg of pure catalase in 1 mL phosphate buffer. H₂O₂ (pro analysi, 30%) was purchased from Sigma-Aldrich (Buchs, Switzerland) and the stock solution is prepared from 13 mL of 30% H₂O₂ which is added to the 100 mL of distilled water. Phosphate buffers KH₂PO₄ and Na₂HPO₄ were from Fisher Chemical (Wien, Austria). Solution with a buffer, pH 7, is prepared from 100 mL of 0.05 mol L⁻¹ KH₂PO₄ and 150 mL of 0.063 mol L⁻¹ Na₂HPO₄ which are added to the 250 mL of distilled water.

Dipotassium trioxohydroxytetrafluorotriborate (K₂[B₃O₃F₄OH]) is water-soluble white powder. Before measurement, the stock solution was prepared by dissolution 20 mg of K₂[B₃O₃F₄OH] in 1 mL phosphate buffer. Tested substance was synthesized in the Laboratory for Physical Chemistry, Department of Chemistry, Faculty of Science, University of Sarajevo, Bosnia and Herzegovina according to the modified method previously described14.

Methods

Manometric method was applied to monitor the enzyme reaction. Measurement of released oxygen in a function of the time was done in a slightly modified apparatus described by Schubert15 at a constant temperature of 37 °C with constant stirring. The total reaction volume of 25 mL, containing a buffer solution with a constant value of catalase and the appropriate amount of K₂[B₃O₃F₄OH] is thermostat, and then entered the appropriate amount of substrate H₂O₂. From the experimental data, the initial velocity V_o was calculated for the appropriate concentration of substrate and K₂[B₃O₃F₄OH].

Results

Determination of K_m and V_max

Catalase decomposes hydrogen peroxide into water and molecular oxygen without the production of free radicals. The kinetic scheme given by Equations (1) and (2) was earlier proposed16 and it was shown that increasing the concentration of H₂O₂ only caused the amplification of this scheme, without modification of the nature of the redox process.

In our study, it was observed that catalase fits the Michaelis–Menten kinetic model and the existence of inhibitor K₂[B₃O₃F₄OH] did not affect the hyperbolic saturation behavior of the enzyme (Figure 1). Lineweaver–Burk plots at different fixed concentrations of K₂[B₃O₃F₄OH] were linear and plotted the family of straight lines that intersect x-axis at the same point (−1/K_m = −0.05, Figure 2). In the absence of inhibitor, the kinetic studies on the activity indicates that the enzyme has a K_m value of 20 mM and V_max value of 0.22 mmoles L⁻¹s⁻¹, which is in good correlation with the common value for K_m and V_max of catalase in literature. In the presence of K₂[B₃O₃F₄OH] from the straight lines intersection of x-axis the K_m for H₂O₂ was found to be constant and equal 20 mM and V_max were variable 0.18, 0.13 and 0.12 mmoles L⁻¹s⁻¹, respecting concentration of K₂[B₃O₃F₄OH].

Figure 1. The Michaelis–Menten plot for catalase: without inhibitor (diamond) and in the presence of 0.4 mM (square); 3.9 mM (triangle) and 69 mM of K₂[B₃O₃F₄OH] (cross).

Figure 2. The Lineweaver–Burk plot for catalase: without inhibitor (diamond) and in the presence of 0.4 mM (square); 3.9 mM (triangle) and 69 mM of K₂[B₃O₃F₄OH] (cross).

The logarithm dose–response plot of percentage inhibition (Figure 3) was studied by varying the concentration of inhibitor K₂[B₃O₃F₄OH] and by measuring and comparing the values of initial rates in the absence and in the presence of inhibitor, at a fixed concentration of [H₂O₂] = 30 mmoles L⁻¹. The activity was expressed in term of IC₅₀ (the concentration required to inhibit the catalase level by 50%) which was calculated from log(dose) curves showing that 50% inhibition of catalase is achieved in the presence 80 mmoles L⁻¹ of K₂[B₃O₃F₄OH] indicating its low impact on catalase activity.

Figure 3. The dose–response plot of initial rates versus logarithm of concentration of inhibitor K₂[B₃O₃F₄OH]. V_o is initial rate in the absence and V_oi is initial rate in the presence of inhibitor. The concentration of [H₂O₂] was fixed at 30 mmoles L⁻¹.

Determination of effect of pH on catalase activity

From the pH assay conducted, it appears that in the absence of inhibitor catalase operates the best at conditions around pH 7.1 and in the presence of K₂[B₃O₃F₄OH] the optimum is around pH 6.2 (Figure 4). It is obvious that the presence of K₂[B₃O₃F₄OH], at a molecular level, affects catalase by altering the state of ionization on the amino acids on the active sites of the enzyme.

Figure 4. Effect of pH on catalase activity: without inhibitor (diamond) and in the presence of 69 mmoles L⁻¹ of K₂[B₃O₃F₄OH] (square).

Determination of activation energy

After the initial rate was determined, the equation: rate = k [H₂O₂]2 was used to determine a reaction rate constant k and ln k. This equation comes from the balanced equation of the overall chemical reaction given by Equations (1) and (2). Then, ln k versus 1/t was plotted and a linear regression found to determine the slopes. Since these slopes are equal to −E_a/R (from linearized Arrhenius equation), it was possible to determine the E_a for the system. The catalase, being a catalyst to the decomposition of H₂O₂, lowers the activation energy to the reaction so it makes the reaction easier to proceed. The uncatalyzed reaction of H₂O₂ decomposition generally has a value of activation energy of 75 kJ mole⁻¹, whereas catalase could lower the value to <8 kJ mole⁻¹17. In our study, in the absence of inhibitor it is determined 11.2 kJ mole⁻¹ and in the presence 69 mmoles L⁻¹ of K₂[B₃O₃F₄OH] it is 37.8 kJ mole⁻¹ showing an increase for 26.6 kJ mole⁻¹.

Discussion and conclusions

Reactive oxygen species (ROS) have been shown to be toxic but also function as signaling molecules. Resistance of tumor cells against intercellular ROS signaling depends on interference through catalase expression on the cell membrane. Intercellular ROS signaling of tumor cells can be restored when H₂O₂ is supplied and catalase is inhibited. These findings define the biochemical basis for specific apoptosis induction in tumor cells and it represents a potential novel approach in tumor prevention and therapy18,19. Previous work20 showed that malignant lung tumors have significantly decreased catalase activity and they suggested that it is plausible that the enzyme catalase could play a role in cancer etiology. Other study suggests that catalase may have multifactorial effects in malignant cells and may decrease tumor progression by modulating the cellular redox state, but enhanced antioxidant capacity of mesothelioma cells also may protect tumor cells against exogenous oxidants21.

Physiologically, catalysts are extremely important, as they greatly facilitate the speed that common chemical reactions occur. Without catalase, essential reactions would take days or months to occur, and toxic H₂O₂ would accumulate quickly in the body. Therefore, it is vital that catalase lower the activation energy of the reaction. At the same time, the presence of 69 mM K₂[B₃O₃F₄OH] raises the activation energy of catalase for 26.6 kJ mole⁻¹ so that the enzyme becomes less efficient. From the pH assay conducted, it was shown that the state of ionization of amino acid is changed and this affects the overall 3D structure of catalase active sites and decreased binding to H₂O₂ substrate occurs by lowering the initial rate. As previously studied, loss of catalase activity is associated with increased susceptibility to oxidative stress22–25. In our study, the inhibitory actions of K₂[B₃O₃F₄OH] were demonstrated only with incubations of mM concentrations of the inhibitor with catalase. These conditions are not possible and representative of the physiological levels and the observed inhibition is unlikely to occur naturally by oral administration of K₂[B₃O₃F₄OH]. Therefore, in case of smaller concentrations of K₂[B₃O₃F₄OH], it could be concluded that it would affect the activity of the catalase and accordingly reduce the formation of ROS. From the other side, in cancer treatment, a mode of action of certain chemotherapeutic agents involves the generation of free radicals to cause cellular damage and necrosis of malignant cell.

Our study shows that the enzyme catalase follows the Michaelis–Menten kinetic model, in the absence and in the presence of inhibitor K₂[B₃O₃F₄OH]. The reaction rate of the catalyst is influenced by factors such as concentration of substrate and inhibitor, temperature and pH. The presence of inhibitor K₂[B₃O₃F₄OH] reduces the maximum rate V_max of enzyme reaction [V_maxI = V_max/(1 + [I]/K_I)] without impact on Michaelis constant K_m [Km is unchanged] suggesting that inhibitor binds equally well to both free catalase and the catalase–H₂O₂ complex. Probably, this binding event occurs exclusively at a site distinct from the precise active site occupied by substrate H₂O₂. Accordingly, although unusual in practice, it seems that K₂[B₃O₃F₄OH] is a classical non-competitive inhibitor and non-competitive inhibition occurs. The proposed mechanism of the K₂[B₃O₃F₄OH] on the inhibition of catalase enzyme activity is presented on Scheme 1.

Scheme 1. The mechanism of the K₂[B₃O₃F₄OH] on the inhibition of catalase enzyme activity.

The inhibitory actions of K₂[B₃O₃F₄OH] were demonstrated only with incubations of millimolar concentrations of the inhibitor with catalase. Therefore, local application of K₂[B₃O₃F₄OH]-containing cream or by its intratumor injection of millimolar concentrations could significantly reduce catalase activity and increase the concentration of H₂O₂ and accordingly produce beneficial effects in tumor tissue alone. Further in vitro and in vivo investigation of antitumor activity of K₂[B₃O₃F₄OH] could explain this hypothesis.

Declaration of interest

This work was partially supported by Ministry of Science, Education and Sports of the Republic of Croatia (Project No. 011-2160547-1330).

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Acknowledgements

We authors thank the Ministry of Science, Education and Sports of the Republic of Croatia for financial support for this study.