Kinetic stabilities of soybean and horseradish peroxidases

doi:10.1016/j.bej.2007.07.019

Biochemical Engineering Journal

Volume 38, Issue 1, 15 January 2008, Pages 110-114

https://doi.org/10.1016/j.bej.2007.07.019 Get rights and content

Abstract

Peroxidases have attractive biocatalytic properties and are used in biosensing and immunoassays. Among various peroxidases, isoenzyme C of horseradish peroxidase (HRP-C) is the most studied, and is also the most commercially used due to its high structural stability. Soybean peroxidase (SBP) and horseradish peroxidase share strikingly similar three-dimensional structures with ∼60% sequence homology. We reported previously, that the conformational and thermal stabilities of SBP are substantially higher than HRP-C. In the present study, we show that the kinetic stability of SBP is much higher than HRP-C as obtained by measuring their unfolding rates at various guanidine hydrochloride (GdnHCl) concentrations. In contrast, the heme-free forms of SBP and HRP-C showed similar kinetic stabilities. We conclude that the higher structural stability of SBP compared to HRP-C stems from the heme binding to the apo protein. Commercial interest of these results is twofold. A cheaper, abundant, better active, and more stable SBP could replace HRP-C. The stability and hence the biocatalytic property of a peroxidase can be improved by suitably engineering the heme active-site that enhances the heme-apo-protein interaction.

Introduction

Peroxidases (donor: H₂O₂, oxidoreductase: EC 1.11.1.7) are heme enzymes catalyzing oxidative reactions that use hydrogen peroxide as an electron acceptor [1]. They have been extensively studied and show many attractive properties for biocatalysis such as wide specificity, high stability in solution and easy accessibility from plant materials. They show potentially interesting applications in a number of fields. The most important application so far is in analytical diagnosis, where they are utilized as a key component of biosensors and immunoassays [2], [3], [4].

Among several plant peroxidases, isoenzyme C of the horseradish perxidase (HRP-C) has been the most widely studied and is the most commercially used peroxidase [5], [6], [7], [8]. It was thought to be the highest stable peroxidase until McEldoon and Dordick [9] showed a higher thermal stability for soybean seed coat peroxidase (SBP); melting temperature (T_m) reported is 90.5 °C for SBP whereas it is 81.5 °C for HRP-C, at pH 8.0 and in 1 mM calcium chloride. Later, we reported a T_m of 86 °C for SBP in absence of any added calcium chloride in the buffer at pH 7.0 [10], which is again much higher than the value reported for HRP-C (74 °C) at identical conditions [11]. Also, we reported for the first time [10] a higher conformational stability for SBP over HRP-C; equilibrium free energy of unfolding [ΔG(H₂O)] of SBP is ∼43 kJ mol⁻¹ [10] as opposed to ∼17 kJ mol⁻¹ of HRP-C [12]. We also found a ∼20fold higher catalytic efficiency for SBP over HRP-C at their pH optima [13]. Both the proteins belong to the same subfamily (Class III) of the plant peroxidase super family and share very similar three-dimensional structures (Fig. 1), amino acid sequence (homology is ∼60%), and catalytic mechanism. They have many structural stabilizing factors in common [1], [14], [15]. They are heme prosthetic group (Fe (III) protoporphyrin IX), four disulfide bonds, two Ca²⁺ ions, and eight glycans.

In any industrial process involving use of an enzyme, it is subjected to varying chemical and physical environments [16]. Choice of the enzyme is therefore dictated by its integrity and functional stability besides operational requirements and economic considerations. Therefore, besides high activity, high conformational and thermal stability requirements, a very high kinetic stability of the enzyme are also essential for their large-scale use. While conformational (thermodynamic) stability is the free energy difference between the unfolded and native states (ΔG(H₂O)) that provides a measure of how much energetically stable the folded state is with respect to unfolded states, kinetic stability on the other hand is the free energy difference between the transition state and native state (activation free energy of unfolding, ΔG*(H₂O)) and hence it is a measure of how much resistant the native state is against external perturbations. In other words, kinetic stability is a measure of the lifetime of an enzyme in its functional state. Kinetic stability can be estimated by measuring the unfolding rate constants at varying concentrations of a denaturant followed by linear extrapolation of the data to obtain the parameters in water [17]. In this paper, we present results on the measurements of the kinetic stabilities of holo and apo forms, of SBP and HRP-C. The kinetic stability of SBP is found to be substantially higher than HRP-C; the activation free energy of unfolding obtained for SBP is ∼96 kJ mol⁻¹ and that obtained for HRP-C is ∼78 kJ mol⁻¹ assuming a rate value of 10⁻⁶ s⁻¹ for the transition state conversion to unfolded state. We further show that the key structural element responsible for the stability as well as differential stabilities of SBP and HRP-C is the heme prosthetic group.

These superior biochemical properties together with the low cost and high abundance of SBP as compared to an expensive and low abundant HRP-C [18] render SBP an ideal candidate to replace HRP-C in industrial and medical applications. Recent research has shown that soluble SBP can be efficiently used as a biocatalyst in the processes such as bio-bleaching of paper dyes [19] and removal of phenol and other aromatic pollutants from waste water [20].

Section snippets

Experimental

SBP (R_Z = A₄₀₃/A₂₈₀ ≈ 0.5), HRP-C (R_Z ≥ 3.0), and GdnHCl were purchased from Sigma Co. SBP was purified by DEAE-Sepharose chromatography [10] to R_Z = 2.8–3.0. Apo-SBP and apo-HRP-C were prepared by the acid-butanone procedure [21]. Concentration of the protein samples prepared in 50 mM phosphate buffer pH 7.0 were determined spectrophotometrically using ɛ₄₀₃ = 94.6 mM⁻¹ cm⁻¹ for SBP [10], ɛ₄₀₃ = 102 mM⁻¹ cm⁻¹ for HRP-C [22], ɛ₂₈₀ = 24 mM⁻¹ cm⁻¹ for apo-SBP [10], and ɛ₂₈₀ = 20 mM⁻¹ cm⁻¹ for apo-HRP-C [23]. The guanidine

Results

Both SBP and HRP-C are resistant to urea [12], [27] and therefore we monitored unfolding in GdnHCl. Fig. 2 represents typical unfolding kinetics of SBP and HRP-C in 7.6 M GdnHCl. All kinetic unfolding curves were fitted to single exponential function in the GdnHCl concentration range of 6.5–8.0 M for SBP, 5.5–8.0 M for HRP-C and 3.4–4.5 M for apo proteins giving rise to the rate constant, k_U, for the unfolding process from native to unfolded protein (N → U). The lower limit of GdnHCl range is set

Discussion

Sequence-structure comparison provides insights into the structural basis of differential stabilities of proteins. A number of factors, such as increased number of hydrogen bonds and salt bridges, optimized packing of hydrophobic core, shortened surface loops, an increased number of prolines, an increase in the number of buried hydrophobic residues, etc. have been proposed for the enhanced stability. It appears that different proteins may use different combinations of structural features to

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SBP demonstrates extraordinary stability and catalytic properties. The melting temperature (at which free energy change is equal to zero) of SBP is 86℃ without Ca2+ at pH 7.0, and rises to 90.5℃ at pH 8.0 with 1 mM Ca2+, hence much higher than those from other peroxidases [1–3]. SBP retains its catalytic activity over a wide pH range (2.4–12) with an optimum pH of 2.4 using veratryl alcohol as substrate, while pH optimum in the range 5.5–6.4 have also been reported using other substrates [3–7].
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¹: Present address: Center for Proteomics, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.

²: Recipient of the TIFR Alumni Association Scholarship for career development, supported by the TIFR endowment fund.

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Short communicationKinetic stabilities of soybean and horseradish peroxidases

Abstract

Introduction

Section snippets

Experimental

Results

Discussion

On the function and mechanism of action of peroxidases

Coord. Chem. Rev.

Development of a colony lift immunoassay to facilitate rapid detection and quantification of Escherichia coli O157:H7 from agar plates and filter monitor membranes

Clin. Diagn. Lab. Immunol.

Drug metabolism biosensors: electrochemical reactivities of cytochrome P450cam immobilised in synthetic vesicular systems

J. Pharm. Biomed. Anal.

Enhanced chemiluminiscence biosensor for the determination of phenolic compounds and hydrogen peroxide

Sensors Actuators B: Chem.

Decolorization of Kraft effluent by free and immobilized lignin peroxidases and horseradish peroxidase

Biotechnol. Lett.

Peroxidase catalyzed removal from coal-conversion waste waters

Science

Peroxidase catalyzed colour removal from bleach plant effluent

Biotechnol. Bioeng.

Horseradish and soybean peroxidases: comparable tools for alternative niches?

Trends Biotechnol.

Unusual thermal stability of soybean peroxidase

Biotechnol. Prog.

Thermal and conformational stability of seed coat soybean peroxidase

Biochemistry

Structural and conformational stability of horseradish peroxidase: effect of temperature and pH

Biochemistry

Conformational states in denaturants of cytochrome c and horseradish peroxidases examined by fluorescence and circular dichroism

Biochemistry

Activity, stability and conformational flexibility of seed coat soybean peroxidase

J. Inorg. Biochem.

Crystal structure of horseradish peroxidase C at 2. 15 A resolution

Nat. Struct. Biol.

Structure of soybean seed coat peroxidase: a plant peroxidase with unusual stability and haem-apoprotein interactions

Protein Sci.

Short communication
Kinetic stabilities of soybean and horseradish peroxidases