Inhibition of Horseradish Peroxidase Activity by Boroxine Derivative, Dipotassium-trioxohydroxytetrafluorotriborate K2[B3O3F4OH]

Ostojic, Jelena; Herenda, Safija; Galijasevic, Semira; Galic, Borivoj; Milos, Mladen

doi:https://doi.org/10.1155/2017/8134350

Journal of Chemistry

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Abstract Introduction Materials and Methods Results and Discussion Conclusion Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2017 | Article ID 8134350 | https://doi.org/10.1155/2017/8134350

Inhibition of Horseradish Peroxidase Activity by Boroxine Derivative, Dipotassium-trioxohydroxytetrafluorotriborate K₂[B₃O₃F₄OH]

Jelena Ostojic,¹Safija Herenda,¹Semira Galijasevic,²Borivoj Galic,¹and Mladen Milos³

Academic Editor: Sevgi Kolaylı

Received14 Nov 2016

Revised17 Feb 2017

Accepted21 Feb 2017

Published08 Mar 2017

Abstract

Recently research shows that horseradish peroxidase, HRP, when combined with other compounds, is highly reactive toward different human tumour cells and that better understanding of catalytic mechanism and inhibition HPR could lead to a new targeted cancer therapy. Thus, the inhibition of HRP activity by dipotassium-trioxohydroxytetrafluorotriborate K₂[B₃O₃F₄OH] was investigated for possible explanation of previously observed antitumour activities of this promising drug. HRP activity was studied under steady-state kinetic conditions by a spectrophotometric method. In the absence of the inhibitor values of = 0.47 mM and = 0.34 mM min⁻¹, respectively, were determined. The hydrogen peroxide H₂O₂ kinetic measurements show a competitive inhibition with the inhibition constant = 2.56 mM. The activation energy values were found to be very similar for both reactions; in the absence of inhibitor activation energy was 17.7 kJ mol⁻¹ and in the presence of inhibitor activation energy was 16.3 kJ mol⁻¹. The values of Arrhenius constants were found to be different; = 4.635 s⁻¹ was measured in the absence of inhibitor while in the presence of inhibitor Arrhenius constant was 1.745 s⁻¹ showing that K₂[B₃O₃F₄OH] initiates conformational change in the structure of the HRP and subsequently reduces its activity.

1. Introduction

Boroxines are the dehydration products of organoboronic acids [1, 2]. Due to their unique electronic structures, these compounds are currently under investigation as possible therapeutics and enzyme inhibitors. The boroxine derivative, dipotassium-trioxohydroxytetrafluorotriborate K₂[B₃O₃F₄OH], has been explored as useful in a treatment of benign and malignant skin changes, such as nevus or skin cancers [3, 4]. First published effects of its bioactivity revealed a potential for inhibition of lymphocytes proliferation and cell growth of basal cell carcinoma culture as well as certain clastogenic potential [5]. Recently it has been confirmed that selected flavonoids may inhibit damage of genetic material in human lymphocytes induced by K₂(B₃O₃F₄OH) [6]. Previous findings of antitumour activity of K₂(B₃O₃F₄OH) in vitro and in vivo on 4T1 mammary carcinoma, B16F10 melanoma, and squamous cell carcinoma SCVII revealed inhibitory effects on cell proliferation in concentration of 1 mM, while concentrations less than 0.1 mM do not significantly affect cell growth in vitro. In vivo, K₂(B₃O₃F₄OH) slows the growth of three tested tumours compared to control regardless of the route of administration (intraperitoneally, intratumour, per oral, or as an ointment) [7]. In recent studies [8–12], the kinetic parameters and inhibition mechanisms of enzymes human carbonic anhydrases and catalase in the presence of some boron-containing anions were investigated, among them K₂[B₃O₃F₄OH], too. It was hypothesized [11] that the local application of K₂[B₃O₃F₄OH]-containing cream, or by its intratumour injection at level of millimolar concentrations, could significantly reduce catalase activity and increase the concentration of hydrogen peroxide H₂O₂ in cell and accordingly produce beneficial effects in tumour tissue alone. In other study [12] it was shown that K₂[B₃O₃F₄OH] is a potent inhibitor of some human carbonic anhydrases with a ranging from 8.0 to 93 μM and it was proposed that K₂[B₃O₃F₄OH] binds to the Zn(II) from active site of enzyme carbonic anhydrase, coordinating to the metal ion monodentately through its boron-OH functionality.

H₂O₂ has many roles in biological systems. One of the major deleterious roles is its involvement in an oxidative damage. During the inflammatory processes, H₂O₂ is produced in large amounts due to activation of macrophages, eosinophils, and neutrophils. In this step, with the help of several enzyme systems such as oxidases or peroxidases free radicals are produced, mainly superoxide radical () that consequently is involved in many reactions causing oxidative damage. H₂O₂ and malfunction of enzymes catalase and different peroxidases play a crucial role in the pathologies of many diseases. Conversely, H₂O₂ is necessary and acts as a second messenger in signal-transduction pathways. Understanding the specific involvement of different inhibitors and their relations between function and enzyme structure may help to design specific inhibitors and prevent diseases in humans, animals, and plants [13]. The family of heme peroxidases as well as HRP catalyse the H₂O₂-dependent oxidation of a wide variety of different substrates [14]. HRP was used for the investigation of inhibitor activity of anti-inflammatory drugs [15]. In other studies it has been found that better understanding the role of HRP and other peroxidases in this process could lead to a new targeted cancer therapy [16–18].

Generally, the ground state (secreted) form of the enzyme, HRP-Fe(III), reacts in a rapid manner with H₂O₂ to form Compound I, two-e⁻ oxidized intermediate possessing, a Fe(IV)=O group, and a resonance-stabilized porphyrin π cation radical. This redox intermediate has a reduction potential of ca. +1 V. Alternatively, Compound I may oxidize multiple substrates through two sequential one e⁻ steps forming Compound II which retains the oxyferryl group but not porphyrin cation radical and HRP-Fe(III), respectively. The conversion of Compound II to HRP-Fe(III) is the rate limiting step of the catalytic cycle of peroxidases. Reaction of excess hydrogen peroxide with the resting state enzyme gives compound III which is the dead end of catalytic process.

Scheme I. Classic catalytic mechanism of HRP is as follows:In this mechanism, AH₂ and are the electron donor substrate and the radical product of one-electron oxidation step, respectively. Electron donor substrate can be a number of inorganic anions, aromatic, and amine compounds.

Since HRP is considered to be as good model for the investigations of kinetic mechanisms and parameters of different enzyme inhibitors, in this paper, we report a study of inhibition impact of K₂[B₃O₃F₄OH] on its activity. This research could help us to better understand peroxidase enzymes activity in general but also the inhibitor properties of K₂[B₃O₃F₄OH] as a new and promising drug.

2. Materials and Methods

2.1. Chemicals

The horseradish peroxidase (EC 1.11.1.7), Guaiacol, and hydrogen peroxide solution (30%) were purchased from Sigma-Aldrich (Buchs, Switzerland) and K₂HPO₄ and KH₂PO₄ from Fisher Chemical (Wien, Austria). K₂[B₃O₃F₄OH] is a boron inorganic derivative prepared by reacting potassium hydrofluoride (KHF₂) with boric acid working in molar ratios of 2 : 3 at room temperature, as reported in the literature [19]. All other compounds used in this study were commercially available as highest purity reagents.

2.2. Assay of HRP Activity

The HRP activity assay was determined by the measurement of Guaiacol peroxidation as described by Chance and Maehly [20]. Guaiacol is oxidized by HRP and the end product, tetraguaiacol (formed from polymerization of the radical product) and is intensely coloured, which makes it very simple to detect [21]. The initial rate of HRP catalytic reaction to oxidize Guaiacol was measured spectrophotometrically (Multiskan GO Microplate spectrophotometer, Thermo Fisher Scientific, USA) by following the increase of absorbance at 470 nm of the reaction mixtures within the first few minutes. The H₂O₂ kinetic measurements were performed at fixed concentration of Guaiacol (1.33 mM) and H₂O₂ concentration varied. The reaction mixture (total volume, 300 μL) consisted of phosphate buffer, pH 6.0 (100 mM), 25 μL of enzyme solution ( = 0.45 nM, assuming the molecular weight of HRP as 44 kDa), 25 μL of Guaiacol, and the proper volume of H₂O₂ to make the above-mentioned concentrations. The reaction mixture also consisted of different amount of K₂[B₃O₃F₄OH] for different series of measurement. In very experiment, inhibitor was then preincubated with enzyme for 5 minutes before each measurement. The reaction was initiated by the addition of H₂O₂.

2.3. Effect of Temperature on HRP Activity

The effect of temperature on the HRP activity was tested at four different temperatures 22, 25, 30, and 37°C under standard conditions for the assays with constant concentrations of [Guaiacol] = 1.33 mM; [H₂O₂] = 0.42 mM; and K₂[B₃O₃F₄OH] = 2 mM, at pH = 6.

2.4. Effect of pH on HRP Activity

The pH effect on the HRP activity was tested under the same working conditions using range of pH 4.5–8.0 in 0.1 M phosphate buffers at fixed concentrations of [Guaiacol] = 1.33 mM and [H₂O₂] = 0.42 mM, in the absence and in the presence of K₂[B₃O₃F₄OH] = 2 mM.

3. Results and Discussion

Guaiacol is necessary for the spectrophotometric monitoring of HRP catalysed degradation of H₂O₂ for enzymes which react with two or more substrates; the Michaelis-Menten equation is inadequate for a full kinetic analysis of such reactions. So, although there are several graphical methods for the determination of kinetic parameters for enzymatic reactions, those are often limited due their low precision and could not reliably determine the type of enzyme inhibition of investigated inhibitor. In present study, the first results for K₂[B₃O₃F₄OH] showed competitive inhibition of HRP. It was unexpected since in our previous investigations K₂[B₃O₃F₄OH] showed noncompetitive inhibition of enzymes catalase and different carbonic anhydrases [11, 12]. Therefore, we decided to carry out a detailed kinetic research by using nonlinear regression [22] and treatment of two-substrate enzyme kinetics [23, 24] in purpose to compare with the results obtained with standard graphical Michaelis-Menten and Lineweaver-Burk methods in treatment by single-substrate enzyme kinetic models [25, 26].

3.1. Determination of and

Some important features of HRP catalytic cycle with Guaiacol as reducing substrate are illustrated in Scheme I. The generation of radical species in two one-electron reduction steps can result in a complex profile of reaction products, including the end product tetraguaiacol (formed from polymerization of the radical product).

Results showed that HRP catalysis fits the Michaelis-Menten kinetic model of reversible enzyme inhibition [25] in the absence and in the presence of K₂[B₃O₃F₄OH] inhibitor. The values of and were determined by nonlinear regression [22] from Lineweaver-Burk plots [26]. In the absence of inhibitor, the nonlinear regression from the best fit of solid line (Figure 1) showed the values = 0.34 mM min⁻¹ and = 0.47 mM. In the presence of different concentrations of K₂[B₃O₃F₄OH], the hydrogen peroxide kinetic measurements showed Lineweaver-Burk plots (Figure 2) and gave set of straight lines that intersect -axis at the same point which determine unique = 0.34 mM min⁻¹. From the straight lines intersection of -axis, the apparent were variable 0.47, 1.35, 1.76, 2.92, and 7.85 mM with respect to concentration of K₂[B₃O₃F₄OH] inhibitor (an example of competitive inhibition is observed).

3.2. Determination of the Inhibitor Constant

The Lineweaver-Burk plots (Figure 2) were used to determine the type of inhibition and evaluation of and , in correlation to concentration of inhibitors. Using single-substrate enzyme reactions and Michaelis-Menten kinetic model, the pattern recognition for these inhibitions was a simple mode for determination of . There is no significant difference between the experimental values and theoretical values of nonlinear regression calculated for particular value of = 2.4 mM (the best fit of solid line in Figure 1).

Since the reported steady-state parameters derived from the Lineweaver-Burk plots give apparent values of Michaelis constant , which depend upon the concentration of guaiacol (1.33 mM) and corresponding concentration of inhibitor, a model of two-substrate enzyme kinetics [23, 24] was applied for the confirmation of values determined by single-substrate model. Therefore, a simple mechanism for the action of K₂[B₃O₃F₄OH] and simplified treatment of two-substrate enzyme kinetics is proposed. In such model the inhibitor K₂[B₃O₃F₄OH] binds to both the native enzyme and Compound II with equal affinity. Further, the HRP/inhibitor complex is completely inactive and does not react with H₂O₂ while the Compound II/inhibitor complex is fully active with respect to Guaiacol oxidation. Essentially, Scheme I can be simplified to two reactions that affect the steady-state oxidation of Guaiacol under the conditions of the current experiments: (1) formation of Compound I and (2) oxidation of Guaiacol by Compound II (Scheme II).

Scheme II. Simplified two-substrate HRP kinetics is as follows: The symbol represents the inhibitor so as not to confuse it with Compound I.

The rate of Guaiacol oxidation by Compound I is about 10 times faster than oxidation by Compound II and only two enzyme species are present at significant concentrations during the steady-state experiments, native HRP and Compound II. In the absence of inhibitor and with H₂O₂ as variable substrate, and are given by the following equations: The ratio of the maximum velocity and Michaelis constant give the most precise value for constants and . Using = 0.34 mM min⁻¹, = 0.47 mM, and = 0.45 nM, the calculated value of rate constant from (3) is = 4.7 × 10⁶ M⁻¹ s⁻¹ and from (4) is = 1.4 × 10⁷ M⁻¹ s⁻¹.

In the presence of inhibitor, the steady-state parameters are given by (3) and by equation for apparent Michaelis constant:In this case, the inhibitor has no effect on the maximum velocity and increases in a linear fashion with inhibitor concentration [], confirming an example of competitive inhibition and consistent with the data shown in Figure 2.

As previously described [27], the evaluation of from these measurements is easily done by plotting the apparent values as a function of inhibitor concentration []. The dependence of the initial velocity () on substrate concentration, [], in the presence of constant inhibitor concentration, may be described by the Michaelis-Menten equation [25] where defined as replaces , the “true” Michaelis constant (obtained in the absence of the inhibitor). Equation (7) may be transformed as follows: Therefore, in pure competitive inhibition systems, a plot versus [] gives a straight line of slope . In our study this plot was linear within experimental error and a weighted linear least-squares analysis gives a slope of = 0.3898 (Figure 3) from which can be calculated to be 2.56 mM. This value is close to the value determined by pattern recognition and so confirming proposed mechanism and competitive inhibitory action of K₂[B₃O₃F₄OH].

In similar studies, HRP was used as a model for studies of cytotoxicities as well as for biologic activities of different kind of compounds [28–30]. The melamine exhibited noncompetitive inhibition of HRP with constant inhibition = 9.5 mM [28] which is slightly weaker than for K₂[B₃O₃F₄OH] determined in our study. In second study [29], the inhibition of intestinal peroxidase activity by nonsteroidal anti-inflammatory drugs indomethacin and acetylsalicylic acid was investigated. Indomethacin showed competitive inhibition with very strong = 32 μM and acetylsalicylic acid exhibited noncompetitive inhibition with = 2.7 mM which is almost equal to for K₂[B₃O₃F₄OH]. Third investigation [30] showed that all investigated uracils interact hydrophobically with HRP heme group, but the measurements showed that only the thiouracil derivatives 2-thiouracil and of 6-n-propyl-2-thiouracil inhibit HRP activity (noncompetitively). Uracil and 6-n-propyluracil do not affect the peroxidase activity.

3.3. Determination of Activation Energy

The activities of HRP and variation of rate constants with temperature changes were examined at four different temperatures 22, 25, 30, and 37°C in the absence and presence K₂[B₃O₃F₄OH] (Figure 4). The Arrhenius constant and activation energy parameters were obtained from the Arrhenius equations [31]:where is the rate constant at corresponding absolute temperature , is preexponential “frequency” factor the called Arrhenius constant, is the activation energy (kJ mol⁻¹), and is the universal gas constant (J/mol).

In our study activation energy values were found to be very similar for both reactions (17.7 kJ mol⁻¹ in the absence and 16.3 kJ mol⁻¹ in the presence of inhibitor), but the values of Arrhenius constants were found to be significantly different, 4.635 and 1.745, respectively, Table 1.

In enzyme reaction, the relative orientation of enzyme at the point of collision with substrate is important. In general the Arrhenius constant is expressed aswhere is “frequency” factor and is a geometrical or steric factor.

Although values of steric factor are difficult to assess in large macromolecules such as enzymes, it can be estimated from observed rate constant with the one in which is assumed to be the same as . In our study, in the absence K₂[B₃O₃F₄OH] it can be assumed that is the same as and in the presence of K₂[B₃O₃F₄OH] the observed different value of rate constant could be due to change of steric factor. This allows us to conclude that K₂[B₃O₃F₄OH] induces a conformational change in HRP molecule affecting steric factor and thus reduces both HRP activity and the rate of reaction, without change of the value of activation energy .

3.4. Effect of pH on HRP Activity

From the performed pH assay, it appears that, in the absence of inhibitor, the HRP operates the best at conditions around pH = 6 and in the presence of K₂[B₃O₃F₄OH] the optimum value is around pH = 7 (Figure 5). It shows that the presence of K₂[B₃O₃F₄OH] affects HRP activity by altering the state of ionization of the amino acids in the active sites of the enzyme.

3.5. Possible Biologic Activity of K₂[B₃O₃F₄OH]

As reactive oxygen species (ROS), H₂O₂ is shown to be toxic but also functions as signalling molecule in cell [32]. Resistance of tumour cells against intercellular signalling depends on interference through peroxidase and catalase expression on the cell membrane. Intercellular ROS signalling of tumour cells can be restored when H₂O₂ is supplied and antioxidant enzymes peroxidase and catalase are inhibited. These findings define the biochemical basis for specific apoptosis induction in tumour cells and it represents a potential novel approach in tumour prevention and therapy [33]. In one study [34] it was showed that malignant lung tumours have significantly decreased antioxidant enzymes activity and authors suggested that it could play a possible role in cancer etiology. In one other study [35] it was suggested that antioxidant enzymes may have multifactorial effects in malignant cells and may decrease tumour progression by modulating the cellular redox state, but enhanced antioxidant capacity of mesothelioma cells also may protect tumour cells against exogenous oxidants. Besides it was shown that loss of catalase activity is associated with increased susceptibility to oxidative stress [36–38]. In previous study [11] it was hypothesized that local application of K₂[B₃O₃F₄OH] containing cream or by its intratumour injection of millimolar concentrations could significantly reduce catalase activity and increase the concentration of H₂O₂ and accordingly produce beneficial effects in tumour tissue alone. In the wake of these considerations, the study of interactions of K₂[B₃O₃F₄OH] with HRP and H₂O₂ is a contribution to the understanding of its previously observed antitumour activity.

4. Conclusion

The K₂[B₃O₃F₄OH] as inhibitor binds to both the native HRP enzyme and Compound II with equal affinity. The HRP/inhibitor complex is completely inactive and does not react with H₂O₂ while the Compound II/inhibitor complex is fully active with respect to Guaiacol oxidation. In relation to hydrogen peroxide kinetic measurements, K₂[B₃O₃F₄OH] has no effect on the maximum velocity and increases in a linear fashion with inhibitor concentration showing an example of pure competitive inhibition. The activation energy has very similar value in the absence and in the presence of inhibitor, suggesting that inhibitor K₂[B₃O₃F₄OH] induces a conformational change in HRP molecule which affects the steric factor and reduces Arrhenius constant and thus reduces both HRP activity and the rate of catalytic reaction, without change of the value of activation energy . It has been shown that pH affects inhibitors properties of K₂[B₃O₃F₄OH]. An optimal effect on HRP activity was around pH = 6 in the absence and pH = 7 in the presence of K₂[B₃O₃F₄OH].

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Acknowledgments

This work has been fully supported by Croatian Science Foundation under Project no. HRZZ-IP-2014-09-6897.

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