Steady-state kinetics of <i>Roystonea regia</i> palm tree peroxidase

doi:10.4236/jbpc.2012.31002

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Vol.3, No.1, 16-28 (2012) Journ al of Biophysical Chemistry

http://dx.doi.org/10.4236/jbpc.2012.31002

Steady-state kinetics of Roystonea regia

palm tree peroxidase

Laura Sánchez Zamorano1, Nazaret Hidalgo Cuadrado1, Patricia Pérez Galende1,

Manuel G. Roig1*, Valery L. Shnyrov2

1Departamento de Química Física, Facultad de Química, Universidad de Salamanca, Salamanca, Spain;

*Corresponding Author: mgr@usal.es

2Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Salamanca, Salamanca, Spain

Received 12 October 2011; revised 20 November 2011; accepted 5 December 2011

ABSTRACT

Royal palm tree peroxidase (RPTP) has been

isolated to homogeneity from leaves of Roys-

tonea regia palm trees. The enz yme puri fication

steps included homogenization, (NH4)SO4 pre-

cipitation, extraction of palm leaf colored com-

pounds and consecutive chromatography on Ph-

enyl-Sepharose, TSK-Gel DEAE-5PW and Super-

dex-200. The novel perox idase w as characterized

as having a molecular weight of 48.2 ± 3.0 kDa

and an isoelectric point pI 5.4 ± 0.1. The enzyme

forms dimers in solution with approximate mo-

lecular weight of 92 ± 2 kDa. Here we invest igated

the steady-state kinetic mechanism of the H2O2-

supported oxidation of different organic substr-

ates by RPTP. The results of the analysis of the

initial rates vs. H2O2 and reducing substrate con-

centrations were seen to be consistent with a

substrate-inhibited Ping-Pong Bi-Bi reaction me-

chanism. The phenomenological approach used

expresses the peroxidase Ping-Pong mechanism

in the form of the Michaelis-Menten equation and

affords an interpretation of the effects in terms of

the kinetic parameters K, , kcat, ,

and of the microscopic rate constants k1

and k3 of the shared three-step peroxidase cata-

lytic cycle. Furthermore, the concentration and

time-dependences and the mechanism of the sui-

cide inactivation of RPTP by hydrogen peroxide

were studied kinetically with guaiacol as co-sub-

strate. The turnover number (r) of H2O2 re quired to

complete the inactivation of the enzyme was 2154

± 100 and t he apparent rate constants of catalysis

185 s–1 and 18 s–1.

mK2

mK22

Keywords: Roystonea regia; Peroxidase;

Steady-State Kinetics; Substrate Inhibition;

Mechanism-Based Inactivatio n Ki ne ti cs ; Hydrogen

Peroxide

1. INTRODUCTION

Peroxidases (EC 1.11.1.7; donor: hydrogen peroxide

oxidoreductase) are widely distributed in the living world

and are involved in many physiological processes. The

oxidation of many biological substances in body fluids

leads to the production of a certain amount of hydrogen

peroxide. Thus, although the function of peroxidases is

often seen mainly in terms of causing the conversion of

toxic H2O2 to H2O, their wider participation in other re-

actions, such as cell wall formation, lignification, the

protection of tissues from pathogenic microorganisms,

suberization, auxin catabolism, defense, stress, etc., should

not be overlooked [1].

Apart from their biological functions, peroxidases are

important as regards many biotechnological applications.

This group of enzymes, especially those of plant origin,

enjoys widespread use as catalysts for phenolic resin

synthesis [2,3] as indicators for food processing and di-

agnostic reagents [4,5], and as additives in bioremedia-

tion [6,7]. Under certain specific conditions, the radicals

formed can break bonds in polymeric materials, destroy-

ing them [8].

Peroxidases reduce hydrogen peroxide and oxidize a

large number of compounds, including phenols, aromatic

amines, thiosanisoles, halide and thyocianate ions, and

fatty acids. Their selectivity with respect to reducing

substrates depends on their specific type [9]. Since they

are able to oxidize a broad variety of organic and inor-

ganic substrates [8], it is somewhat difficult to determine

which substrates are physiologically relevant for plant

peroxidases.

Peroxidases have been identified throughout the plant

kingdom, but that from horseradish root (HRP), and in

particular the slightly basic C isoenzyme (HRPC), is the

one that has attracted the most attention. Despite this,

recently more data have become available regarding

L. S. Zamorano et al. / Journal of Biophysical Chemistry 3 (2012) 16-28 17

peroxidases from other plants, such as peanut [10], bar-

ley [11], tea [12], Arabidopsis thaliana [13], and palm

trees [14-18].

The shared three-step catalytic cycle of peroxidases,

involving different intermediate enzyme forms, is known

as the Poulos-Kraut mechanism [19,20]. Catalysis is ini-

tiated by the binding of H2O2 to the high-spin ferric haem

iron of resting peroxidase, followed by heterolytic cleav-

age of the peroxide oxygen-oxygen bond under the in-

fluence of highly conserved histidine and arginine resi-

dues at the active site [21]. The haem undergoes a two-

electron oxidation, forming an intermediate (Compound I)

that contains an oxyferryl species (Fe(IV)=O) and a por-

phyrin π-cation radical. As the co-product of the reac-

tion, a water molecule is generated. Completion of the

catalytic cycle usually involves two successive single-

electron transfers from separate reducing substrate mole-

cules to the enzyme. The first reduction, of the porphyrin

π-cation radical in Compound I, yields a second enzyme

intermediate, Compound II, which retains the iron in the

oxyferryl state [22]. Under steady-state conditions, the

reduction of Compound II, to recover the ferric enzyme,

is often rate-limiting. The extremely reactive free radi-

cals released from the catalytic cycle often condense

spontaneously, giving rise to polymers.

The peroxidase cycle is generally considered irre-

versible. Nevertheless, it is unquestionable that adsorp-

tion complexes between the enzyme and its substrates

exist physically [23]. The microscopic constants govern-

ing the equilibrium between aromatic compounds and

peroxidase have been estimated. Even though the co-

substrates (donor or H2O2) in the enzyme modulate each

other’s affinity, it is possible for the mechanism to pro-

ceed via random binding. This observation, together with

retain special kinetic features [24], supports the notion

that there is no need for the peroxide to bind to the en-

zyme prior to donor adsorption.

Here we investigated the kinetic mechanism of the

H2O2-supported oxidation of different organic substrates

by means of a novel plant peroxidase from the Roystonea

regia palm tree (RPTP).

Among the broad variety of organic and inorganic

substrates of peroxidases, in the present work we ex-

plored six organic chromogenic substrates: three pheno-

lics, guaiacol (2-metoxiphenol), catechol (2-hydroxyphe-

nol) and ferulic acid (3-(4-hydroxy-3-methoxyphenyl)-2-

propenoic acid), and ABTS (2,2’-azino-bis(3-ethylbenz-

thiazoline-6-sulphonic acid), often used as a reference

substrate, o-dianisidine and o-phenylendiamine, the latter

three suitable for use in ELISA procedures that employ

peroxidase conjugates [25-28].

Since these oxidation reactions exhibit Michaelis-

Menten saturation kinetics with respect to both substrates,

the chromogenic substrate and H2O2, the system was

amenable to steady-state kinetic experiments, which

were used to deduce the kinetic mechanism of the reac-

tion following methodologies established for two-sub-

strate enzyme systems [29]. The results of the initial-rate

and inhibition studies carried out here indicate that the

H2O2-supported oxidation of different organic substrates

catalyzed by this peroxidase proceeds via a Ping-Pong

Bi-Bi mechanism mediated by the oxidized enzyme in-

termediate Compounds I and II.

Palm peroxidase catalyzes the oxidation reactions of a

large variety of reducing substrates, using H2O2 as oxi-

dizing agent [19,30,31]. In the absence of reducing sub-

strates, excess H2O2 leads to the inactivation of the en-

zyme, in this case acting as a suicide substrate of per-

oxidase and being irreversibly bound to its active site [32,

33]. Despite this, it has been suggested that HRP inacti-

vation by hydrogen peroxide would be due to the forma-

tion of one or several non-active enzyme products, pro-

bably through the formation of Compound III (peroxyl-

FeIII porphyrin) [34,35].

The oxidative inactivation of peroxidases is mecha-

nism-based. The molecular mechanism driving this hy-

drogen peroxide-mediated inactivation is extraordinarily

complex because an array of reactions can occur subse-

quent to the reaction of the haem iron with the hydrop-

eroxide. Regardless of the differences among the per-

oxidases, a common inactivation mechanism involving

several stages can be proposed. In the absence of sub-

strate, or when they are exposed to high hydrogen per-

oxide concentrations, peroxidases show the kinetic be-

havior of suicide inactivation, in which hydrogen perox-

ide is the suicide substrate that converts Compound II

into a highly reactive peroxy-iron(III) porphyrin free-

radical termed Compound III [36]. Compound III does

not form part of the peroxidase cycle, but is produced

under excessive exposure of protonated Compound II to

oxidative species in a reaction that is partially mediated

by free superoxide radicals [37].

Despite representing different structural groups, ki-

netic models for the hydrogen peroxide-mediated inacti-

vation of horseradish peroxidase (HRP) [33], ascorbate

peroxidase (APX) [38], peroxidase from the Royal Palm

Tree (RPTP) [16], microperoxidase-11 [39] and Chamae-

rops excelsa peroxidase (CEP) [40] are similar in the

sense that they are time-dependent and exhibit saturation

kinetics. From the inactivation stoichiometry, it has been

concluded that for APX only 2.5 molecules of hydrogen

peroxide are required per active site for the inactivation

form to be generated [38], in contrast to the 265 mole-

cules required for HRP [33]. This difference is due to the

low catalytic activity of HRP, which is absent in APX

[41]. For APX peroxidase, inactivation is correlated with

enzyme bleaching, suggesting haem destruction [38].

Another factor in this difference is the glycosylation of

L. S. Zamorano et al. / Journal of Biophysical Chemistry 3 (2012) 16-28

the enzyme, which seems to be important in protecting

the enzyme from inactivation [33].

2. EXPERIMENTAL

2.1. Materials

Analytical or extra-pure grade polyethyleneglycol (PEG),

guaiacol, catechol, ferulic acid, ABTS, o-dianisidine, o-

phenylendiamine, ammonium sulfate, sodium phosphate

and Tris-HCl were purchased from Sigma Chemical Co.

(St. Louis MO, USA) and were used without further pu-

rification. H2O2 was from Merck (Darmstadt, Germany).

Superdex-200 columns and Phenyl-Sepharose CL-4B

columns were from GE Helthcare Bio-Sciences AB (Up-

psala, Sweden). TSK-Gel DEAE-5PW was purchased

from Tosoh Co. (Tokyo, Japan). Cellulose membrane

tubing for dialysis (avg. flat width 3.0 in) was purchased

from Sigma Chemical Co.; slide A-lyzer dialysis cas-

settes (extra-strength, 3 - 12 mL capacity, 10.000 MWCO)

were form Pierce Biotechnology, Inc. (Rockford, IL,

USA) and filter devices (Amicon Ultra Cellulose 10.000

MWCO, 15 mL capacity) were from Millipore Corp.

(Billerica, MA, USA). All other reagents were of the

highest purity available. The water used for preparing the

solutions was double-distilled and then subject to a de-

ionisation process.

2.2. Enzyme Purification

RPTP was purified from palm tree Roystonea regia as

described [15,17] but with some modifications. The pu-

rity of the RPTP was determined by SDS-PAGE as de-

scribed by Fairbanks et al. [42] on a Bio-Rad Minigel

device using a flat block with 12% polyacrylamide con-

centration; by gel filtration, which was performed using a

Superdex 200 10/30 HR column in an FPLC Amersham

Äkta System; and by UV-visible spectrophotometry (RZ

= A403/A280 = 2.8 - 3.0). Analytical isoelectrofocusing

was performed on a Mini IEF cell model 111 (Bio-Rad

Laboratories, Hercules, CA, USA) using Ampholine

PAG-plates, pH 3.5 - 9.5 (GE Healthcare Biosxiencies

AB, Upsala, Sweden). The electrophoretic conditions

and Silver Staining Kit Protein were as recommended by

manufacturer. The standards used were from a broad-

range pI calibration kit (4.45 - 9.6) from Bio-Rad Labo-

ratories (Hercules, CA, USA.).

2.3. Enzymatic Activity of RPTP

The initial rates of appearance of the products of oxi-

dation of different substrates (guaiacol, catechol, ferulic

acid, ABTS, o-dianisidine, o-phenylendiamine) due to

the catalytic action of RPTP in the presence of H2O2

were measured by electronic absorption spectroscopy at

the characteristic wavelengths of such products (470, 295,

318, 414, 420 and 445 nm, respectively) [30]. The reac-

tions, initiated by the addition of RPTP, were performed at

25˚C in 20.0 - 30.0 mM universal buffer, containing vari-

able concentrations of the reducing substrate at fixed H2O2

concentration and viceversa. The reactions were carried

out at the optimal pH for each substrate, 6.9 for guaiacol,

3.5 for catechol, 4.0 for ferulic acid, 3.0 for ABTS and 6.0

for o-dianisidine and o-phenylendiamine [43].

The concentration of peroxidase was measured spec-

trophotometrically at 403 nm, using the experimentally

determined extinction coefficient value of 60.8 ± 2.3

mM–1·cm–1 for the protein monomer [43].

To determine the microscopic rate constants and other

kinetic parameters for the oxidation of the substrates by

RPTP in the presence of H2O2, the mathematical treat-

ment of Morales and Ros-Barceló [44] was applied. The

initial reaction rates were obtained from the kinetic runs

and fitted vs. substrate concentration, at fixed H2O2 con-

centration, and viceversa, according to the generally ac-

cepted two-substrate Ping-Pong mechanism for the per-

oxidases [19,20].

2.4. Inactivation Experiments

RPTP was inactivated at 25˚C in 10 mL incubations of

universal buffer, pH 6.5, containing a fixed amount of

the enzyme (136 nM). The reactions were started by the

addition of H2O2 (over a range of concentrations). At

specified time intervals, 5 - 20 μL aliquots of the incuba-

tion mixtures were transferred to cuvettes containing 2

mL of an assay mixture composed of 18 mM guaiacol

and 4.9 mM H2O2. Peroxidase activity was measured by

the increase in absorbance at 470 nm [30]; neither the

substrate nor the RPTP present in the assay mixture in-

terfered with the measurement. A minimum of three in-

cubation assays for each peroxide concentration were

performed. The residual enzymatic activity (AR) was

taken as the enzymatic activity remaining (At) as a per-

centage of the initial activity (Ao). The residual peroxi-

dase activity was assayed at different times and after 24 h

of incubation.

3. RESULTS AND DISCUSSION

3.1. Enzyme Purification

RPTP was purified to homegeneity with a high yield

from palm tree Roystonea regia leaves. The purification

steps and their efficiencies are summarized in Table 1.

Purified peroxidase migrated in SDS-PAGE as a single

band corresponding to a molecular weight of 48.2 ± 3.0

kDa [43]. The retention time of the elution of protein

from the size-exclusion column indicates that the enzyme

forms dimers in solution with approximate molecular

eight of 92 ± 2 kDa [37]. Thin-layer IEF confirmed the w

L. S. Zamorano et al. / Journal of Biophysical Chemistry 3 (2012) 16-28

Table 1. Purification of RPTP.

Procedure Volume (ml) Protein (mg) Total activity (U) Specific activity (U/mg) Purification factor Yield (%)

1 Homogenate 9980 68,862 1,297,422 18.84 1 100

2 PEG + (NH4)2SO4 5400 11,502 1,293,635 112.47 6 99.7

3 Phenyl-Sepharose 383 498 845,825 1698.78 90 65.2

4 DEAE-Toyopearl 99.5 158 581,665 3676.65 195 44.8

5 Superdex 200 42 67 438,626 6568.22 350 33.8

apparent homogeneity of the protein and enabled us to

estimate the pI of the protein to be around 5.4 ± 0.1 [43].

3.2. Steady-State Rate Equation and Kinetic

Parameters of RPTP-Catalyzed

Oxidation Reaction

In a two-substrate enzyme system, two general mecha-

nisms are possible for the substrate-enzyme interaction:

namely, a sequential mechanism or a Ping-Pong mecha-

nism. In the former, both substrates combine with the

enzyme to form a ternary complex before catalysis oc-

curs. The substrates can combine with the enzyme either

in a random fashion (Random Bi Bi) or in an obligatory

order (Ordered Bi Bi) to form the ternary complex. The

products thus formed can therefore be released in an or-

dered or random fashion. In a Ping-Pong mechanism, a

ternary substrate-enzyme complex is not formed. The

first substrate in a Ping-Pong Bi Bi mechanism combines

with the enzyme to form a substituted enzyme interme-

diate, with the ensuing release of the first product. The

second substrate then interacts with the substituted en-

zyme intermediate to form the second product and re-

generate the native enzyme. Ping-Pong and sequential

mechanisms can be differentiated by steady-state kinetic

analysis of the reaction using the procedures described

by Cleland [45,46].

Figure 1. Primary double-reciprocal plot of the initial rate

of ferulic acid oxidation as a function of hydrogen peroxide

concentration at fixed ferulic acid concentrations (0.023

mM (●), 0.045 mM (○), 0.053 mM (■), 0.076 mM (□)).

The insets show the secondary plots of 1/V vs. 1/[ ferulic

acid] (A) and of 1/K vs. 1/[ferulic acid] (B). See text for

other experimental conditions.

shown). Similar trends towards parallel lines in double-

reciprocal plots were also observed at different pHs for

guaiacol, catechol, ABTS, o-dianisidine and o-phenylen-

diamine. The obtained trends towards such linear parallel

plots point to a Ping-Pong Bi Bi mechanism involving

two independent enzyme forms (i.e. enzyme forms sepa-

rated by an irreversible step).

The kinetic mechanism of the H2O2-assisted RPTP-

catalyzed oxidation of AH2 reducing substrates was in-

vestigated using initial-rate measurements, in which the

concentrations of both substrates, -H2O2 and AH2- were

varied systematically and the results were analyzed as-

suming steady-state conditions. The initial rates, v, as a

function of hydrogen peroxide or the AH2 concentration,

were fitted to the Michaelis-Menten rate equation (Eq.A2

of the Appendix) by an iterative process [47].

Thus, upon representing the intercept (1/V) of the

above lines and the inverse of the K parameter vs. the

reciprocal of the fixed substrate concentration, linear

relationships are obtained (insets in Figure 1 for the fer-

ulic acid case). The values of , , Vmax and

kcat, shown in Table 2, were calculated from the slopes

and intercepts of the corresponding linear fittings of data

following Eqs.A6 and A7.

At pH 4.0, double-reciprocal plots of initial steady-

state rates of ferulic acid oxidation vs. hydrogen peroxide

concentration (0.1 - 2.4 mM), at fixed reducing substrate

ferulic acid concentrations, afforded a set of approxi-

mately parallel lines, as shown in Figure 1. A similar

graphic behaviour of the double-reciprocal plots of the

data was obtained upon studying the effect of ferulic acid

concentrations on the initial rates of the oxidation reac-

tion at different fixed H2O2 concentrations (data not

The highest turnover number, kcat, of RPTP was found

for the substrate ferulic acid, followed by catechol,

ABTS, guaiacol, o-phenylenediamine and o-dianisidine.

The highest affinity of the enzyme (1/Km) was for o-

dianisidine, followed by ABTS, ferulic acid, o-phenyle-

nediamine, guaiacol and catechol. However, the highest

specificity constant, or catalytic efficacy of the enzyme

(kcat/Km), was seen for ferulic acid, followed by ABTS, o-

OPEN A CCESS

L. S. Zamorano et al. / Journal of Biophysical Chemistry 3 (2012) 16-28

Table 2. Kinetic parameters obtained for the H2O2-mediated oxidation of substrates by RPTP. See text for experimental conditions.

Substrate Km (H2O2)

(M)

Km (AH2)

(M)

Vmax

(M·s–1)

[Eo]

(10–10 M)

kcat

(s–1) kcat/Km (H2O2)

(M–1·s–1)

kcat/Km (AH2)

(M–1·s–1)

(µM–1·s–1)

Guaiacol 2.7 × 10–3 15.2 × 10–3 1.2 × 10–6 5.61 2.1 × 1037.8 × 105 1.4 × 105 0.33 0.08

ABTS 1.8 × 10–3 4.9 × 10–4 2.0 × 10–6 6.05 3.3 × 1031.8 × 106 6.7 × 105 1.30 4.80

Ferulic acid 1.9 × 10–3 5.4 × 10–4 4.8 × 10–6 8.91 5.4 × 1042.8 × 107 1.0 × 108 1.82 6.96

o-Dianisidine 7.0 × 10-4 1.0 × 10–4 1.2 × 10–7 2.97 4.0 × 1025.7 × 105 4.0 × 106 0.25 1.91

o-Phenilendiamine 2.2 × 10–3 1.4 × 10–3 7.8 × 10–7 10.2 7.6 × 1023.5 × 105 5.5 × 105 0.23 0.26

Catechol 3.5 × 10–3 8.8 × 10–2 2.8 × 10–5 35.0 8.0 × 1032.3 × 106 9.0 × 104 0.40 0.02

dianisidine, o-phenylenediamine, guaiacol and catechol.

Similar reactivities for these substrates have been found

for African [30] and Chamaerops excelsa [48] palm tree

peroxidases.

3.3. Microscopic Rate Constants

Peroxidases catalyze the oxidation of AH2 organic

substrates, using H2O2 (or other peroxides) as an electron

acceptor in a three-step catalytic cycle involving differ-

ent intermediate enzyme forms [19,20]:

22 2

EHOEIHO

 (1)

EI AHEII AH

k

 (2)

EIIAHEHO AH

k

 (3)

where E is the native enzyme. The monoelectronic oxi-

dation of the native state E affords an intermediate state

termed EI (Eq.1). EI is responsible for the oxidation of

the electron-donor substrate (AH2), accepting one proton

and one electron and generating its free radical (AH



together with another enzyme state, designated EII (Eq.2).

Finally, EII is reduced by a second molecule of sub-

strate (Eq.3), giving rise to a second free radical (AH•).

The microscopic constant k1 (the constant of EI forma-

tion) indicates the reactivity of the enzyme towards hy-

drogen peroxide, and k3 (the constant of EII reduction)

represents the reactivity of the enzyme towards the re-

ducing substrate.

With a view to calculating the microscopic constants

(k1 and k3) of the oxidation of the substrates by RPTP, the

oxidation rates of the substrates were fitted for each

concentration of AH2 and H2O2, assuming the steady-

state approach and considering that k2 > k3.

As may be seen in the Appendix, double-reciprocal

plots (1/v vs. 1/[H2O2]) allowed us to calculate the A and

B values for each AH2 concentration. Figure 2 shows the

plot of A vs. B (Eq.A8) for four ferulic acid concentra-

tions. From this straight line it is possible to calculate the

value of k1 (formation constant of Compound I) for per-

oxidase-mediated ferulic acid oxidation. The value ob-

tained, as well as those obtained for guaiacol, catechol,

ABTS, o-dianisidine and o-phenylendiamine, is shown in

Table 2.

As shown in the Appendix, double reciprocal plots

(1/v vs. 1/[AH2]) allowed us to calculate the A and B

values (Eq.A9) for each H2O2 concentration. Figure 3

shows the plot of A vs. B values for three H2O2 concen-

trations during the oxidation of ferulic acid. With this

plot it is possible to calculate the value of k3 (the forma-

values (Eq.A9) for each H2O2 concentration. Figure 3

Figure 2. Secondary plot of parameters A

(nmol·s–1) vs. B (mM) obtained by varying H2O2

concentra- tions for four ferulic acid concentrations

(0.023, 0.045, 0.053, 0.076 mM). See text for other

expe- rimental conditions.

Figure 3. Secondary plot of parameters A

(nmol·s–1) vs. B (mM) obtained by varying ferulic

acid con- centrations for five H2O2 concentrations

(0.1, 0.15, 0.30, 0.50, 1.00 mM). See text for other

experi- mental conditions.

L. S. Zamorano et al. / Journal of Biophysical Chemistry 3 (2012) 16-28 21

shows the plot of A vs. B values for three H2O2 concen-

trations during the oxidation of ferulic acid. With this

plot it is possible to calculate the value of k3 (the forma-

tion constant of Compound II) for peroxidase-mediated

ferulic acid oxidation. The value obtained, as well as

those obtained for guaiacol, catechol, ABTS, o-dianis-

idine and o-phenylendiamine, is also listed in Table 2.

These A vs. B plots allowed us to calculate the real re-

action constants (ki) from steady-state measurements of

the oxidation rate, avoiding their dependence on the sub-

strate concentration [20].

From the rate constant values (ki) shown in Table 2, it

may be deduced that the RPTP is capable of oxidizing

phenolic and aromatic amine substrates. The data ob-

tained show that the most reactive substrate for RPTP

was ferulic acid, followed by ABTS, the aromatic amines

o-dianisidine and o-phenylendiamine, guaiacol and cate-

chol being the least reactive substrates. The high reactiv-

ity of ABTS must be due to its greater number of H-bond

acceptor atoms, because the highest values of reactivity

constants are seen for substrates with the most acceptor

H-bonds (www.chemicalregister.com). ABTS has ten H-

bond acceptor sites; ferulic acid and o-dianisidine four,

and o-phenylendiamine, guaiacol and catechol two. In

respect of H-bond donor sites—ABTS, ferulic acid, o-

dianisidine, o-phenylendiamine, catechol—each has two

H-bond donor sites and guaiacol only one.

Similar studies addressing kinetic parameters and mi-

croscopic rate constants carried out with African [30] and

Chamaerops excelsa [48] palm tree peroxidases have

shown that these enzymes exhibit greater reactivity to-

wards ferulic acid and ABTS, followed by the aromatic

amines o-dianisidine, o-phenylendiamine and, finally, by

phenolic substrates with one or two hydroxyl groups in

their chemical structures. In contrast, both soybean and

peanut peroxidases are more reactive towards guaiacol

than towards amines [30,49]. Horseradish and tobacco

peroxidases have been reported to be equally reactive

towards guaiacol and o-dianisidine and about 10 - 15

times less reactive towards o-phenylendiamine [43].

Commonly substrate specificity studies of peroxidases

are conducted with only one substrate present, apart from

H2O2, in the reaction mixture at a given time; i.e. without

any alternative substrates able to undergo the same reac-

tion. This is because the presence of competing sub-

strates tends to complicate the analysis, without provid-

ing much more information than would be obtained by

studying the substrates separately. However, this implies

an important difference between experimental practice

and the physiological conditions under which enzymes

usually exist. Thus, most enzymes are not perfectly spe-

cific for a single substrate and must often select between

several that are available simultaneously. Therefore, to

be physiologically meaningful enzyme specificity must

be defined in terms of how well the enzyme can dis-

criminate between the substrates present in the same re-

action mixture. This does not mean that it cannot be de-

termined from the kinetic parameters of the enzyme for

separate substrates, but it does mean that these parame-

ters need to be interpreted correctly and not on a casual

basis [50].

3.4. Substrate Inhibition

In order to further check the kinetic mechanism of the

substrate oxidation reactions catalyzed by RPTP, inhibi-

tion studies were carried out. One of the characteristic

features of Ping-Pong reaction mechanisms is the occur-

rence of competitive substrate inhibition by both sub-

strates [29].

In the Ping-Pong reaction mechanism, since the three

forms of the enzyme—E, CoI and CoII—are so similar, it

is reasonable to expect AH2 to have some affinity for E

as well as CoI and CoII and, if the active sites in CoI and

CoII are not too full for the adsorption of H2O2, for H2O2

to show some affinity for CoI and CoII [51]. In peroxi-

dases, reports have been made of the formation of a

non-productive, or dead-end, complex between AH2 and

E and the reaction of high concentrations of H2O2 with

CoI, affording H2O2 and O2 [52].

As stated in the Appendix, following the reciprocal of

Eq.A13, plots of 1/v vs. 1/[H2O2] at fixed [AH2] should

be linear and should intersect on the y axis. This graphi-

cal behaviour was observed at fixed ferulic acid inhibit-

tory concentrations (Figure 4).

In this sense, Figure 4 (inset) shows this linear plot for

ferulic acid. For the other substrates studied, a similar

KN (mM·s)

Figure 4. Primary double-reciprocal plot of the initial rate

of ferulic acid oxidation as a function of hydrogen peroxide

concentration at fixed ferulic acid inhibitory concentrations

(0.095 mM (■), 0.104 mM (□), 0.113 mM (○), 0.128 mM

(●)). The inset shows the secondary plot of K/V vs. ferulic

acid concentration. See text for other experimental condi-

tions.

L. S. Zamorano et al. / Journal of Biophysical Chemistry 3 (2012) 16-28

degree of substrate competitive inhibition was found

(data not shown).

Alternatively, at fixed inhibitory values of H2O2 con-

centration, the v vs. [AH2] data analysis provided a series

of alternative equations similar to Eqs.A13-A16.

Accordingly, the corresponding plots of 1/v vs. 1/[AH2]

at fixed [H2O2] are also linear and intersect on the y axis

and the plots of K/V vs. [H2O2] should also be linear.

This graphic behaviour was obtained for ferulic acid at

fixed [H2O2] (Figure 5) and also for the rest of substrates

studied (data not shown).

Thus, competitive substrate inhibition was observed in

the H2O2-assisted RPTP-catalyzed oxidation reactions

for both substrates, as would be expected for a Ping-

Pong reaction mechanism. The corresponding inhibition

constants— and —obtained, for the case of

ferulic acid, were 1.24 × 10–3 M and 2.7 × 10–5 M, re-

spectively.

In light of the above, the combined initial-rate and

substrate inhibition results exclude an ordered or random

sequential reaction mechanism, and are only consistent

with a Ping-Pong Bi Bi mechanism according to the no-

tation of Cleland [29] as the minimal kinetic model for

H2O2-assisted RPTP-catalyzed substrate oxidation reac-

tions.

Ping-Pong reaction kinetics has also been observed for

several other peroxidase-catalyzed oxidations mediated

by Compound I. The results of initial-rate studies of the

hydrogen peroxide-supported oxidation of guaiacol by

turnip peroxidase [53] and of the oxidation of ferrocyto-

chrome c catalyzed by horseradish peroxidase [54] and

yeast cytochrome c peroxidase [55] are consistent with a

Ping-Pong mechanism. Initial-rate kinetic studies of the

KN (mM·s)

Figure 5. Primary double-reciprocal plot of the initial rate

of ferulic acid oxidation as a function of ferulic acid con-

centration at fixed hydrogen peroxide inhibitory concentra-

tions (1.5 mM (■), 2.0 mM (○), 3.0 mM (●)). The inset

shows the secondary plot of K/V vs. H2O2 concentration.

See text for other experimental conditions.

oxidation of ferrocytochrome c by Pseudomonas aerugi-

nosa cytochrome c peroxidase yielded intersecting plots,

which were initially interpreted as indicating a sequential

reaction mechanism [56]. However, subsequent studies

demonstrated that the intersecting plots arose from the

formation of an inactive hydrogen peroxide-enzyme com-

plex, and the mechanism of the reaction was reinter-

preted to be of the modified Ping-Pong type [57]. It has

recently been reported that Chamaerops excelsa palm

tree peroxidase also exhibits a Ping-Pong Bi Bi mecha-

nism for the H2O2-assisted catalyzed oxidation reactions

of guaiacol, ABTS, o-dianisidine and o-phenylendiamine

[48].

3.5. Partitioning Ratio (r) for the Inactivation

of RPTP by H2O2

Using plots of the percent residual activity for each

substrate against the [peroxide]/[enzyme] ratio, the ratio

required in each case for 100% inactivation can be ob-

tained from the intercept of the fitted line. From this

value, the partitioning ratio can be calculated using the

following equation:







A1rRPT



P

(4)

where AR is the residual activity; At and Ao are the activi-

ties at time (t) (end of the reaction) and zero respectively;

(r) is the partitioning ratio or inactivation turnover num-

ber (number of catalytic cycles given by enzymes before

their inactivation), and [H2O2] and [RPTP] are the initial

concentrations of H2O2 and enzyme [33].

Figure 6 shows the plots of the percent residual activ-

ity against the [H2O2]/[RPTP] ratio for the guaiacol sub-

strate. In light of the consumption of two moles of H2O2

in each catalytic cycle (one mole for the formation of

Compound I and another for inactivation or catalysis)

[58-60], the (r) value, calculated from the corresponding

fitting of the data to the first linear section of the curve

(Figure 6), was 2154 ± 100.

3.6. Kinetics of Inactivation by H2O2

In the absence of reducing substrate, the class I, II and

III peroxidases studied to date undergo suicide inactiva-

tion by H2O2. The inactivation kinetics shows profiles

that differ for each enzyme. The inactivation kinetics of

RPTP by hydrogen peroxide, at a range of different con-

centrations, using guaiacol as co-substrate, is biphasic,

with a rapid inactivation step in the first 10 - 15 min,

followed by a longer-lasting, slow step (data not shown).

The same biphasic behaviour has been reported for the

inactivation by H2O2 of HRP [32,33,61] and melon per-

oxidase [62]. Other peroxidases studied to date also

L. S. Zamorano et al. / Journal of Biophysical Chemistry 3 (2012) 16-28 23

Figure 6. Sensitivity to inactivation of RPTP at differ-

ent [H2O2]/[RPT] molar ratios. RPTP was incubated

with molar excesses of peroxide in universal buffer (30

mM, pH 6.5) and when the reaction was complete (24

h incubation), the percentage residual activities were

measured with guaiacol at a fixed concentration of the

enzyme (136 nM).

exhibit different types of behaviour [33,48].

Then, the biphasic behaviour of data of residual per-

oxidase activity versus time could be fitted to a sum of

exponentials:

ob ob

Aae be

k



 tk

(5)

Consequently, following Eq.6, a plot of the logarithms

of the percentage residual activities (lnAR) against time

afforded straight lines for each segment of time (data not

shown), with slopes equivalent to the observed rate con-

stants of the inactivation (kob, kob′) [38].







ln t

Ak (6)

The inactivation of RPTP by H2O2 clearly showed

saturation kinetics, as seen from the hyperbolic curves

fitted to the plot of kob against [H2O2] for each segment

of time (Figure 7).

Then, the observed first-order rate constants of inacti-

vation (kob) can be fitted to the following equation:







app

inact22

ob app

2

(7)

where , a first-order inactivation rate constant, and

app

inact

app

, an inhibitor-binding constant, were obtained from

the corresponding fitting of the data by a linear regres-

sion model [63].

In the absence of reducing substrates and at high H2O2

concentrations, RPTP is inactivated in a time- and H2O2

concentration-dependent process, exhibiting suicide or

mechanism-based inactivation kinetics. Similar types of

behaviour have been reported by other authors [16,32,33,

Figure 7. Biphasic inactivation kinetics of

RPTP by H2O2. Plot of kob (first-order in-

activation rate constant) against H2O2 con-

centration for the first and rapid inactiva-

tion step (A) and for the longer-lasting

slow step (B).

61,64] in studies addressing H2O2-mediated inactivation

under identical experimental conditions of temperature,

pH, and the concentrations of peroxidase and H2O2.

Nevertheless there are, differences in the shapes of the

inactivation curves, in the magnitudes and rates of inac-

tivation at specific H2O2 concentrations, and in the

maximum rate of inactivation () as compared with

the results reported here.

app

inact

The inactivation process involves the participation of

two pathways, one reversible and other irreversible,

which may or may not function independently of each

other and whose individual contribution to the overall

inactivation process seems to be dependent upon the

H2O2 concentration. For enzyme inactivation, a second

molecule of hydrogen peroxide would be required; not

for the inactivation per se but for the formation of Com-

pound III. Once Compound III has been formed, a frac-

tion of that population would be transformed into an in-

active species. Another interpretation would be that once

a molecule of Compound III has been formed, it has a

certain probability of decaying into an inactive species

instead of decaying into an active one that would again

become engaged in the catalytic cycle.

In the case of Compound I, several authors [32,33,64]

have suggested the existence of a partitioning between

the two pathways. Their model, based on studies at high

H2O2 concentrations, invokes a further partitioning be-

tween these two inactivation pathways and suggests the

existence of a catalytic reaction in which H2O2 would be

consumed with relatively little harm to the enzyme [64].

The model is actually a simplification of the full ki-

netic approach developed previously by Arnao et al.,

L. S. Zamorano et al. / Journal of Biophysical Chemistry 3 (2012) 16-28

Table 3. Apparent kinetic constants calculated for the inactivation of RPTP (*, #) and CEP variants by H2O2 at 25˚C.

Substrate r

app

inact

k (s–1) app

(mM)app

(M–1)app

cat

k (s–1)app app

cat I

kK (s–1·M–1) app app

inactI

kK (s–1·M–1)

Guaiacol* 2154 0.086 25.63 0.039 185.2 723 3.36

Guaiacol# 2154 0.0083 66.03 0.015 17.9 270 1.26

ABTS 4844 3.24 845 1.18 15694 18573 3.83

Guaiacol 3711 3.85 204 4.90 14287 70034 18.87

o-Dianisidine 951 1.27 39.6 25.2 1209 30530 32.07

o-Phenilendiamine 2147 1.79 87.1 11.5 3839 44076 20.55

*Rapid inactivation of RPTP. #Slow inactivation of RPTP.

4. CONCLUSIONS [63], although it does offer a reasonable approximation

that would be suitable for comparative purposes, as re-

quired here. Double-reciprocal plots of rate (or kob) of

inhibition vs. inhibitor concentration data are often used

to explore the formation of complexes between inhibitors

and enzymes [64-66]. Here we used these linear least-

squares fits of the data (data not shown) to determine the

relationship between the different enzymes and H2O2.

For each variant we obtained the apparent values of the

inactivation rate constant () and the dissociation

constant (

app

inact

app

) of the [Compound I.H2O2] complex and,

together with the already determined inactivation turn-

over numbers (r) (see above), the values of the catalytic

rate constant (), using the relationship:

app

cat

As results of the analysis of the initial rates vs. H2O2

and reducing substrate concentrations carried out, the

proposed steady-state kinetic model of the H2O2-sup-

ported oxidation of different organic substrates by RPTP

is a substrate-inhibited Ping-Pong Bi Bi reaction mecha-

nism. The phenomenological approach used expresses

the peroxidase Ping-Pong mechanism in the form of the

Michaelis-Menten equation and affords an interpretation

of the effects in terms of the kinetic parameters ,

, kcat, , and of the microscopic rate

constants k1 and k3 of the shared three-step peroxidase

catalytic cycle. This substrate-inhibited Ping-Pong Bi Bi

reaction mechanism has been also proposed for peroxi-

dase from Chamaerops excelsa palm tree [48].

K22

app

cat

app

inact

 (8) The kinetics of inactivation of RPTP in the oxidation

of guaiacol by hydrogen peroxide shows suicide inacti-

vation behavior similar to that of most classical peroxi-

dases [32,33,38,41]. The model used in these experi-

ments [38] provides satisfactory parameters for the inac-

tivation kinetics by hydrogen peroxide, showing the high

capacity of the enzyme to act the substrate at a turnover

of molecules up to 2154 and exhibits an apparent rate

constant of catalysis of 185 s–1 and 18 s–1. These values

may indicate that RPTP exhibits a good performance

against inactivation by hydrogen peroxide, opening the

possibility of further studies related to the mechanisms of

exchange of hydrogen peroxide in peroxidases and

pointing, besides other studies [17], that RPTP acts as a

very robust enzyme.

These findings, together with the corresponding to the

affinity of the enzyme for the inhibitor (app

1K) and to

the efficiencies of catalysis (appapp

cat I

kK) and inactivation

(app app

inact I

kK), are summarized in Table 3, which—for

comparative purposes—also includes the results of the

inactivation of Chamaerops excelsa peroxidase [40].

CEP is the most active of all currently known peroxi-

dases, suggesting that the active site of the enzyme has

evolved not only to improve catalytic efficiency but also

to prevent inactivation by the highly reactive H2O2 sub-

strate.

The accessibility of the substrate to the reducing bind-

ing site in the haem pocket and its affinity for the product

are important for enzyme activity. Substrate specificity

may be modified by changes that affect the reducing

binding site [67] and it may be possible to further adjust

the specificity and level of activity of RPTP by judicious

changes in this region. The glycosylation of RPTP also

appears to be significant in protecting the enzyme from

inactivation.

5. ACKNOWLEDGEMENTS

Funding from Consejeria de Educación (projects SA129A07 and

SA052A10-2) and Consejeria de Agricultura y Ganaderia (Project

SA06000) of the Regional Government of Castilla and León (Junta de

Castilla y León, Spain) is acknowledged.

RPTP shows good resistance to inactivation by hy-

drogen peroxide, opening the possibility of further stud-

ies related to the mechanisms of exchange of hydrogen

peroxide in peroxidases and suggesting, as in other stud-

ies [17], that RPTP acts as a very robust enzyme.

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APPENDIX where V and K parameters are as follows:



max

V1A



The rate equation for an enzyme-catalyzed Ping-Pong

reaction with two substrates, H2O2 and AH2, in the ab-

sence of products and at non-inhibitory substrate con-

centrations, is given by

(A3)



H

(A4)









 

22 2

max2 22

HO AH

m2m2222

VHOAH

vAHH OH OAHKK



(A1) Thus plots of 1/v vs. 1/[H2O2] are linear and parallel at

different fixed [AH2] since:

which can be cast in the form of a rectangular hyperbola

for fixed values of [AH2]:



vVVHO

 (A5)







VHO

vHO

 (A2) Furthermore, the reciprocals of Eqs.A3 and A4 are

L. S. Zamorano et al. / Journal of Biophysical Chemistry 3 (2012) 16-28

given by:



max max2

11 1

VVV AH

 (A6)



22 22

HO HO

11 1

KKK

 (A7)

For non-inhibitory concentrations of H2O2, the initial

rates of the substrate oxidation by peroxidases, following

the three-step catalytic cycle [19,20], can be fitted to the

following equation:







AHO

vBHO

 (A8)

where A = 2[E]k3[AH2] and B = (k3/k1)[AH2]. Double-

reciprocal plots (1/v vs. 1/[H2O2]) allowed us to calculate

A and B values for each AH2 concentration

Similarly, the dependence of v on [AH2] may be written

as:







AAH

vBAH

 (A9)

where A = 2[E]k1[H2O2] and B = (k1/k3)[H2O2]. Double

reciprocal plots (1/v vs. 1/[AH2]) allowed us to calculate

the A and B values for each H2O2 concentration

From Eq.A8, the catalytic efficacy for the utilization of

H2O2 would be given by:









cat

31 2

2AH 2

K (A10)

while the catalytic efficacy for the utilization of the

substrate AH2 would be given by:









122

cat

13 22

2HO2

K

(A11)

Consequently, the reactivity of the enzyme with

hydrogen peroxide is determined by the value of the

constant k1. However, its reactivity with the reducing

substrate is determined by the constant k3.

The occurrence of competitive substrate inhibition by

both substrates in the reaction mechanism means that in

the denominator of the rate Eq.A1, the





AHK



term can be multiplied by



1AH and the





HOK2

term by







22 IS

1HO K where

KAH

and are the dissociation constants of AH2

from EAH2 and of H2O2 from CoI H2O2 and/or CoII

H2O2 complexes, respectively, (Eq.A12), and double

competitive substrate inhibition would be exhibited when

AH2 and H2O2 are varied. Thus, the corresponding rate

equation would be Eq.A12.

At fixed inhibitory values of the AH2 concentration,

the v vs. [H2O2] data were fitted to the following rate

equation of competitive inhibition:







max2 2

VHO

vAH

1HKK











(A13)

For competitive inhibition, the K and V of Eq.A2 are

defined by



max

V1AK

H

(A14)









22 2

HO AH

1AH





(A15)

Thus, following the reciprocal of Eq.A13, plots of 1/v

vs. 1/[H2O2] at fixed [AH2] should be linear and should

intersect on the y axis.

Furthermore, the plots of K/V vs. [AH2] should be lin-

ear because





HO m

SI max

VV V

 2

(A16)

Alternatively, at fixed inhibitory values of H2O2 con-

centration, the v vs. [AH2] data analysis should provide a

series of alternative equations similar to Eqs.A13- A16.



















222 222

max2 22

H OAHAHH O

m22SIm2222SI222

VHOAH

vAH1AHH O1HOH OAHKKK K

 (A12)