Vol.3, No.1, 16-28 (2012) Journ al of Biophysical Chemistry
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
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.
Keywords: Roystonea regia; Peroxidase;
Steady-State Kinetics; Substrate Inhibition;
Mechanism-Based Inactivatio n Ki ne ti cs ; Hydrogen
Peroxidases (EC; 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 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
Copyright © 2012 SciRes. OPEN ACCESS
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
Copyright © 2012 SciRes. OPEN ACCES S
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].
2.1. 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.
3.1. 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
Copyright © 2012 SciRes. OPEN ACCESS
L. S. Zamorano et al. / Journal of Biophysical Chemistry 3 (2012) 16-28
Copyright © 2012 SciRes.
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-
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)
Km (AH2)
(10–10 M)
(s–1) kcat/Km (H2O2)
kcat/Km (AH2)
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
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
 (1)
 (2)
 (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.
Copyright © 2012 SciRes. OPEN ACCESS
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-
Copyright © 2012 SciRes. OPEN ACCES S
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-
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-
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
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:
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
Copyright © 2012 SciRes. OPEN ACCESS
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
ob ob
Aae be
 tk
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:
ob app
where , a first-order inactivation rate constant, and
, 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.
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.,
Copyright © 2012 SciRes. OPEN ACCES S
L. S. Zamorano et al. / Journal of Biophysical Chemistry 3 (2012) 16-28
Copyright © 2012 SciRes.
Table 3. Apparent kinetic constants calculated for the inactivation of RPTP (*, #) and CEP variants by H2O2 at 25˚C.
Substrate r
k (s–1) app
k (s–1)app app
cat I
kK (s–1·M–1) app app
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 (
) 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:
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].
(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-
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
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:
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
 
22 2
max2 22
(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]:
 (A5)
(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
 (A6)
22 22
11 1
 (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:
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
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:
31 2
2AH 2
K (A10)
while the catalytic efficacy for the utilization of the
substrate AH2 would be given by:
13 22
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
term can be multiplied by
1AH and the
term by
22 IS
1HO K where
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
For competitive inhibition, the K and V of Eq.A2 are
defined by
22 2
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
 2
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
 (A12)
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