Cadmium(II)-stimulated enzyme activation of Arabidopsis thaliana phytochelatin synthase 1

Article abstract of DOI:10.1016/j.jinorgbio.2010.09.011

Phytochelatin (PC), a class of heavy metal-binding peptides, is synthesized from the tripeptide glutathione (GSH) and/or previously synthesized PC in a reaction mediated by PC synthase (PCS). In the present study, the PC production rate catalyzed by recombinant Arabidopsis PCS1 (rAtPCS1) in the presence of a constant free Cd(II) level increased steadily and the kinetic parameters were approximated using a substituted-enzyme mechanism in which GSH and bis(glutathionato)cadmium acted as co-substrates. In contrast, the PC production rate as a function of GSH concentration at a constant total Cd(II) concentration reached a maximum, which shifted toward higher GSH concentrations as the concentration of Cd(II) was increased. These observations are consistent with the suggestion that rAtPCS1 possesses a Cd(II) binding site where Cd(II) binds to activate the enzyme. The affinity constant, optimized using a one-site mathematical model, successfully simulated the experimental data for the assay system using lower concentrations of Cd(II) (5 or 10 μM) but not for the assay using higher concentrations (50 or 500 μM), where a sigmoidal increase in PCS activity was evident. Furthermore, the PCS activity determined at a constant GSH concentration as a function of Cd(II) concentration also reached a maximum. These findings demonstrate that rAtPCS1 also possesses a second Cd(II) binding site where Cd(II) binds to induce an inhibitory effect. A two-site mathematical model was applied successfully to account for the observed phenomena, supporting the suggestion that rAtPCS1 possesses two Cd(II) binding sites.

Full text of DOI:10.1016/j.jinorgbio.2010.09.011

Journal of Inorganic Biochemistry 105 (2011) 111–117
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Cadmium(II)-stimulated enzyme activation of Arabidopsis thaliana
phytochelatin synthase 1
Shinya Ogawa, Takahiro Yoshidomi, Etsuro Yoshimura ⁎
Department of Applied Biological Chemistry, School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 August 2010
Received in revised form 25 September 2010
Accepted 27 September 2010
Available online 23 November 2010
Phytochelatin (PC), a class of heavy metal-binding peptides, is synthesized from the tripeptide glutathione
(
GSH) and/or previously synthesized PC in a reaction mediated by PC synthase (PCS). In the present study, the
PC production rate catalyzed by recombinant Arabidopsis PCS1 (rAtPCS1) in the presence of a constant free Cd
II) level increased steadily and the kinetic parameters were approximated using a substituted-enzyme
(
mechanism in which GSH and bis(glutathionato)cadmium acted as co-substrates. In contrast, the PC
production rate as a function of GSH concentration at a constant total Cd(II) concentration reached a
maximum, which shifted toward higher GSH concentrations as the concentration of Cd(II) was increased.
These observations are consistent with the suggestion that rAtPCS1 possesses a Cd(II) binding site where Cd
(II) binds to activate the enzyme. The affinity constant, optimized using a one-site mathematical model,
successfully simulated the experimental data for the assay system using lower concentrations of Cd(II) (5 or
10 μM) but not for the assay using higher concentrations (50 or 500 μM), where a sigmoidal increase in PCS
activity was evident. Furthermore, the PCS activity determined at a constant GSH concentration as a function
of Cd(II) concentration also reached a maximum. These findings demonstrate that rAtPCS1 also possesses a
second Cd(II) binding site where Cd(II) binds to induce an inhibitory effect. A two-site mathematical model
was applied successfully to account for the observed phenomena, supporting the suggestion that rAtPCS1
possesses two Cd(II) binding sites.
Keywords:
Arabidopsis thaliana
Cadmium
Phytochelatin
Glutathion
Phytochelatin synthase
©
2010 Elsevier Inc. All rights reserved.
1
. Introduction
more effectively [6]. Metals and metalloids belonging to groups 11–15
in the fourth, fifth, and sixth periods of the periodic table can activate
PCS-mediated PC synthesis [7]. As PCS is expressed constitutively in
cells, the metal ion dependency of PC synthesis represents a self-
terminating reaction in which the activator metal ions are captured by
the reaction product.
The enzymatic mechanism of action for PCS in relation to
activation by metal ions has yet to be resolved. Adding Cd(II) to a
solution containing GSH and PCS from Silene cucubalus initiates PC
synthesis, and the supplementing the solution with Cd(II) chelators,
Phytochelatins (PCs) are a family of peptides with the general
n n
structure (γ-Glu-Cys) -Gly (PC ), where n≥2. The peptides, desig-
nated as cadystin, were first identified in the fission yeast Schizo-
saccharomyces pombe grown in Cd(II)-containing medium [1]. Shortly
after the initial identification, the peptides were detected in cells of
the higher plant Rauvolfia serpentina in suspension culture [2]. To
date, PCs have been identified in higher plants, algae, and some fungi
exposed to toxic levels of heavy metal ions [3]. PCs play a role in
detoxification by sequestering heavy metal ions, although other roles
in Zn(II) homeostasis and the long-distance transport of Zn(II) have
been suggested [4].
n
such as uncomplexed PC mixtures or ethylenediaminetetraacetic
acid, immediately terminates PC synthesis, suggesting that PCS is
activated by metal ion binding [5]. Equilibrium dialysis of AtPCS1-
FLAG indicated seven Cd(II) binding sites in the protein with a
dissociation constant of 0.54 μM [8]. However, under standard PCS
assay conditions, in which GSH is present at a much higher level than
Cd(II), the level of free Cd(II) ions is extremely low; using the reported
equilibrium binding constants for Cd(II) and GSH, the free Cd(II)
concentration can be predicted, which has been calculated as
The synthesis of PC is catalyzed by phytochelatin synthase (PCS)
using the tripeptide glutathione (γ-Glu-Cys-Gly; GSH) and/or
previously synthesized PC as a substrate [5]. PCS, a γ-glutamylcystei-
nyl transpeptidase (EC 2.3.2.15), mediates a dipeptidyl transfer
reaction in the presence of heavy metals, leading to the formation of
PC
n
with a greater number of γ-glutamylcysteinyl units, which has
−
13
increased affinity for heavy metals and the capacity to sequester them
6.638×10
M when Cd(II) and GSH are present at total concentra-
tions of 25 μM and 3.3 mM, respectively, at pH 8.0 [8]. Thus, the
concentration of free Cd(II) ions is too low to account for the binding
of Cd(II) ions to AtPCS1-FLAG [8]. Kinetic analysis of PC synthesis
mediated by AtPCS1-FLAG has indicated a reaction process consistent
⁎
Corresponding author. Tel.: +81 3 5841 5153; fax: +81 3 5841 8027.
E-mail address: ayoshim@mail.ecc.u-tokyo.ac.jp (E. Yoshimura).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.09.011

112
S. Ogawa et al. / Journal of Inorganic Biochemistry 105 (2011) 111–117
with a bisubstrate-substituted mechanism, whereby a GSH molecule
and a Cd(II)-G complex act as co-substrates and the enzyme is
acid (TFA). The mixture was diluted with 0.1% TFA in which 0.5 μM
desglycyl PC was added as an internal standard. PC concentrations
2
2
already in an active form without binding to Cd(II) [8]. Furthermore, it
has been inferred from the facility of AtPCS1 for the synthesis of S-
alkylated PCs from S-alkylated GSH in the absence of added heavy
metal ions that GSH derivatives with thiol groups blocked either
through heavy metal thiolate formation or S-alkylation are solely
contingent on PC synthesis [8]. However, peptide scanning of PCS
from wheat Triticum aestivum L. (TaPCS1) and the fission yeast S.
pombe (SpPCS) revealed that Cd(II) binds to the proteins in the
presence of GSH at a concentration of 1 mM [9]. In addition, Cd(II)-
dependent stabilization of PCS has been reported for the enzyme
produced by the primitive red alga Cyanidioschyzon merolae, support-
ing the suggestion that Cd(II) binds to the enzyme [10]. Therefore, no
definitive conclusion has been reached regarding the metal activation
mechanism of PC synthesis.
were determined by HPLC using detection based on the dequenching
of Cu(I)-BCS complexes [13,14]. The peptides were separated on an
octadecylsilane column (4.6 mm ϕ×150 mm, TSKgel ODS-80™;
Tosoh, Tokyo, Japan) with a mobile phase consisting of 5% acetoni-
trile/0.1% TFA using a pump (PU-1580; Jasco, Tokyo, Japan) at a flow
−
1
rate of 1 mL min . Using another pump (PU-980; Jasco) at a flow
−
1
rate of 1 mL min , the eluent was merged with a post-column
solution containing 50 mM CHES-NaOH (pH 10.0), 0.2 μM CuSO
4
,
0.5 μM BCS, and 5 μM ascorbic acid. The merged solution was passed
through a mixing coil, and the fluorescence intensity at excitation and
emission wavelengths of 280 and 395 nm, respectively, was moni-
tored using an FP-920 fluorescence detector (Jasco). During HPLC
analyses, the post-column solution was bubbled continuously with a
stream of He. The integrated peak area was used to quantify the PC
level after calibration with chemically synthesized PC (Hayashi Kasei,
2
. Materials and methods
Osaka, Japan). PCS activity was expressed as the amount of PC
synthesized by 1 mg of protein per minute, because the synthesis of
PC with n greater than 2 was negligible.
2
2
.1. Heterologous expression and purification of N-terminal
n
hexahistidinyl-tagged Arabidopsis thaliana PCS1
2.3. PCS activity assays in the presence of a constant level of free Cd(II)
We used a recombinant Arabidopsis thaliana PCS1 tagged with
hexahistidine at its N-terminus (rAtPCS1). The AtPCS1 DNA sequence
was amplified using PrimeSTAR HS DNA polymerase (TakaraBio,
Shiga, Japan) and a plasmid containing the AtPCS1 gene (Riken
BioResource Center, Saitama, Japan) [11,12] was used as a template.
The forward and reverse PCR primers were 5′-GAGAGCTAGCATGGC-
TATGGCGAGTTTATATCG-3′ (NheI site underlined) and 5′-GAGA-
GAATTCCTAATAGGCAGGAGCAGCGAGATC-3′ (EcoRI site underlined),
respectively. The resultant DNA fragment was transferred to the pET-
PCS activity was determined at various GSH concentrations while
keeping the free Cd(II) concentration constant. For this purpose, the
total Cd(II) and GSH concentrations were changed concomitantly
from the initial values of 5 μM and 10 mM, respectively, in such a way
2
−2
−1
that the ratio of [Cd(II)]
t
to [G]
t
was maintained at 5×10
M
. The
M
−
10
concentration of free Cd(II) ions was calculated to be 1.22×10
under these conditions (Supplementary 1 and 2).
2
8b expression vector (Novagen, Madison, WI) as an in-frame fusion
2.4. Determination of parameters
with a plasmid-encoded hexahistidine tag sequence, and the plasmid
was used to transform Escherichia coli XL-1 Blue (Stratagene, La Jolla,
CA). Following the confirmation of successful cloning by sequencing
with T7 primers, the E. coli BL21 Rosetta (DE3) pLysS strain (Novagen)
was transformed with the construct.
Non-linear least squares analysis was performed using the Origin
8.1 program (OriginLab, Northampton, MA).
3. Results
E. coli cells carrying rAtPCS1 were pre-cultured in 200 mL of Luria-
Bertani broth at 25 °C. After reaching an optical density at 600 nm of
3.1. PC synthesis activity of rAtPCS1 as a function of GSH concentration
0
.1–0.8, isopropyl 1-thio-β-D-galactoside was added to 40 μM, and the
culture was incubated for a further 6 h to express the recombinant
protein. The cells were collected by centrifugation at 8000×g for
The PCS activity of rAtPCS1 as a function of the concentration of
GSH supplemented in the assay solution is shown in Fig. 1. In the
presence of 10 μM Cd(II) (Fig. 1, closed squares), activity increased
and reached the maximum rate with an increase in GSH concentration
1
5 min and then suspended in an extraction solution (25 mM Tris–
HCl, pH 8.0, 400 mM NaCl, 10% glycerol, 1 mM 2-mercaptoethanol,
.1% Tween 20, and 1 mM phenylmethylsulfonyl fluoride). The cells
0
were disrupted by sonication, and the supernatant was obtained by
centrifugation at 13,000×g for 10 min at 4 °C. The recombinant
protein was purified on a Ni2 affinity column by fast-performance
liquid chromatography (Pharmacia LKB, Uppsala, Sweden). The
supernatant (20 mL) was applied to a 1-mL HisTrap™ HP column
+
(
GE Healthcare Life Sciences, Piscataway, NJ) equilibrated with
binding buffer (20 mM HEPES-NaOH, pH 7.4, 500 mM NaCl, 1 mM
-mercaptoethanol, and 40 mM imidazole), the column was washed
2
with 10 mL of binding buffer, and then the sample was eluted with a
linear gradient of 40–250 mM imidazole in binding buffer over
2
0 min. The rAtPCS1 eluted at 150 mM imidazole. Analysis of the
eluate by SDS-PAGE revealed a single band with a molecular mass of
5
6.5 kDa, confirming the homogeneity of the preparation.
2
.2. PCS activity assays
The reaction mixture (100 μL) contained 100 ng/mL rAtPCS1,
0 mM 2-mercaptoethanol, 100 ng/mL bovine serum albumin, and
various concentrations of GSH and CdSO in 200 mM HEPES-NaOH
4
buffer (pH 8.0). The mixture was incubated at 35 °C for 15 min, and
Fig. 1. Effects of GSH concentration on the PCS activity of rAtPCS1 in assay solution
containing total Cd(II) concentrations of 1 (closed triangles), 5 (closed circles), and
1
10 μM (closed squares). The solid lines (a, b, and c) represent simulated activity using
the one-site model for the assay solutions containing 1, 5, and 10 μM Cd(II),
respectively, where the Vmax and KE1 values listed in Table 1 were used.
the reaction was terminated by adding 25 μL of 10% trifluoroacetic

S. Ogawa et al. / Journal of Inorganic Biochemistry 105 (2011) 111–117
113
up to 15 mM, and then decreased with further increases in GSH
concentration. A similar trend was observed for the enzyme assay
system in which 1 or 5 μM Cd(II) was added (Fig. 1, closed circles and
triangles, respectively). It was also evident that the maximal activity
occurred at higher GSH concentrations with an increase in Cd(II)
concentration. Thus, the maximal activity was attained at GSH
concentrations around 10 and 15 mM when the enzyme was assayed
in the presence of 5 and 10 μM total Cd(II), respectively. Although the
GSH concentration at which the maximal activity attained may be
obscure in the enzyme assay system with 1 μM total Cd(II), it occurred
at a concentration less than or equal to 5 mM. These phenomena still
held for the assay solution containing higher levels of total Cd(II). As
shown in Fig. 2, maximal PCS activity occurred at 30 and 60 mM GSH
for the assay solution containing 50 μM (closed squares) and 500 μM
Cd(II) (closed circles), respectively. In addition, a sigmoidal increase
in activity was observed at GSH concentrations below 20 mM for the
assay system containing 50 and 500 μM Cd(II), a prominent feature for
the system at high levels of Cd(II).
3
.2. PC synthesis activity of rAtPCS1 as a function of the total Cd(II)
concentration
The PCS activity of rAtPCS1 as a function of Cd(II) added to the
assay solution is illustrated in Fig. 3. At a constant GSH concentration
of 5 mM, PCS activity increased with increasing Cd(II) concentration
up to 4 μM, and then decreased sharply (Fig. 3A). A similar activity
profile was obtained for the solution containing 20 mM GSH, where
the maximal activity was attained at 60 μM Cd(II) (Fig. 3B). These
results indicated that rAtPCS1 has an optimum Cd(II) concentration
for PCS activity, which shifts toward a higher Cd(II) concentration in
the presence of higher levels of GSH.
3
.3. PC synthesis activity of rAtPCS1 as a function of GSH concentration
at a constant free Cd(II) concentration
PCS activity at a constant free Cd(II) concentration of 1.22×
0− M was determined as a function of total GSH concentration.
10
Fig. 3. Effects of total Cd(II) concentration on the PCS activity of rAtPCS1 in the presence
of 5 mM (A) and 20 mM (B) GSH. The inset in (A) is an expansion of the figure at Cd(II)
concentrations from 0 to 40 μM. The solid lines represent the simulated activity, where
the Vmax, KE1, and KE2 values listed in Table 2 were used.
1
The activity increased steadily with total GSH concentration up to
0 mM (Fig. 4A). A Hanes plot of [G] /activity versus [G] showed a
5
t
t
straight line at GSH concentrations greater than 25 mM, whereas the
plot deviated upward at GSH concentrations below 25 mM (Fig. 4B).
These findings indicated that the PCS synthesis reaction can display
Michaelis–Menten-type kinetics only at GSH concentrations above
2
4
4
5 mM.
. Discussion
.1. PCS activity of rAtPCS1 as a function of GSH
Although a definitive conclusion has not been reached regarding
the mechanisms of metal ion activation of PC synthesis, enzyme
acylation by a γ-glutamylcysteinyl group has been clearly demon-
strated [15]. The thiol group of Cys-56 in AtPCS1 has been identified as
an acylation site, which undergoes a reaction with GSH in a Cd(II)-
independent manner [15]. The γ-glutamylcysteinyl group on an acyl
intermediate is transferred to an acceptor GSH or PCn − 1 molecule in
PC
γ-glutamylcysteinyl dipeptide transfer. In the case of PC
for example, the γ-glutamylcysteinyl group on PCS is transferred to a
Cd(II)-G , in which the enzyme is already in an active form with no
n
synthesis. Two distinct mechanisms have been proposed for the
2
synthesis,
2
need for the direct binding of metals [8]. The alternative mechanism is
that Cd(II) is required to bind PCS to activate it for the transfer of the
dipeptide to an acceptor molecule such as GSH or PCn − 1 [5].
The PCS activity of rAtPCS1 determined at a constant total Cd(II)
concentration demonstrated the maximum activity with respect to
GSH concentration (Figs. 1 and 2). In addition, with an increase in the
Fig. 2. Effects of GSH concentration on the PCS activity of rAtPCS1 in the assay solution
containing total Cd(II) concentrations of 50 (closed squares) and 500 μM (closed
circles). The solid lines (a and b) represent simulated activity using the two-site model
for the assay solutions containing 50 and 500 μM Cd(II), respectively, where the Vmax
E1, and KE2 values listed in Table 3 were used.
,
K

114
S. Ogawa et al. / Journal of Inorganic Biochemistry 105 (2011) 111–117
concentration was attributable to a lower free Cd(II) level. These
findings clearly demonstrated that AtPCS1 activation requires Cd(II)
binding.
A proposed substituted-enzyme mechanism [8] in which the
enzyme is in an active form without being bound by Cd(II) and the
2
GSH and Cd(II)-G molecules act as co-substrates is in conflict with
the declining PCS activity at higher GSH concentrations at a constant
total Cd(II) concentration, because an increase in GSH concentration
at a constant total Cd(II) concentration will also elevate the free GSH
2
levels and Cd-G levels. To make such a mechanism consistent with
our observations, it would be necessary to hypothesize a GSH binding
site on the enzyme that exerts an inhibitory effect. The steady increase
in PCS activity with GSH concentration in the presence of a constant
free Cd(II) level may be attributable to an increased concentration of
Cd(II)-G
2
, the effect of which may surpass the inhibitory effect with
increased
regard to PCS activity, as the concentration of Cd(II)-G
2
concomitant with that of GSH in the assay system (Fig. 4). However,
this possibility can be excluded because the PCS activity at a constant
free Cd(II) level approximately followed Michaelis–Menten-type
kinetics at higher GSH concentrations. These observations indicated
that the Cd(II)-G binding site was almost completely occupied by the
2
complex and also suggested that there is no such GSH binding site on
the enzyme, as discussed later.
2
4.2. Kinetic analysis of PC production mediated by rAtPCS1 at a constant
free Cd(II) level
The acylation of the enzyme by a γ-glutamylcysteinyl group occurs
in a Cd(II)-independent manner [15], which leads to substituted-
enzyme mechanisms as a PC synthesis mechanism. In general, using
the maximal reaction rate, Vmax, and the enzyme reaction rate, v, a
substituted-enzyme mechanism can be expressed as:
Vmax½Aꢀ½Bꢀ
v =
ð1Þ
KmB½Aꢀ + KmA½Bꢀ + ½Aꢀ½Bꢀ
Fig. 4. PCS activity of rAtPCS1 as a function of GSH concentration at a constant free Cd
(
based on a substituted-enzyme mechanism, in which the Michaelis constants for GSH
where A and B denote substrates that bound first and second to the
enzyme, respectively, and KmA and KmB represent the Michaelis'
constants for substrates A and B, respectively [16]. From Eq. (1), it
appears that if the same substances act as substrates (i.e., A = B),
then the reaction will follow simple Michaelis–Menten-type kinetics.
However, this does not appear to be the case for PCS-catalyzed PC
II) level of 1.22×10− M (A). The simulated PCS activity indicated by a solid curve is
10
and Cd(II)-G
2
used were 18.0 mM and 5.14 μM, respectively. A Hanes plot, [G] /activity
t
/activity and [G] at GSH
t
versus [G] , represents a linear relationship between [G]
t
t
concentrations greater than 25 mM, whereas the plots deviate upward from the linear
relationship (B). This observation indicated that the PC synthesis reaction can be
approximated by Michaelis–Menten-type kinetics only for GSH concentrations greater
than 25 mM, as can be deduced from Eq. (2).
t t
synthesis, because the Hanes plot of [G] /activity versus [G] yielded a
non-linear relationship at GSH concentrations below 25 mM (Fig. 4B).
From these results, together with the observation that enzyme
acylation occurred even in the absence of Cd(II) addition, it is likely that
total Cd(II) concentration, the maximal activity shifted to a higher
GSH concentration (Figs. 1 and 2). These observations were consistent
with the notion that rAtPCS1 possesses a Cd binding site and that the
binding of Cd(II) to this site activates the enzyme. The increase in PCS
activity up to the GSH concentration at which the maximal rate is
attained is due to the increasing concentration of the substrate GSH. In
contrast, the suppression of enzyme activity at GSH concentrations
greater than that at which the maximal activity is attained is
attributable to a reduction in the level of activated (Cd(II)-bound)
rAtPCS1. An increase in the concentration of GSH would reduce the
level of free Cd(II), resulting in a reduction in the amount of rAtPCS1
bound by Cd(II).
a GSH molecule and a Cd(II)-G complex react as substrates binding to
2
rAtPCS1 first and second, respectively, as suggested by Vatamaniuk et al.
[8]. As the levels of Cd(II)-G complexes can be approximated by the total
2
Cd(II) concentrations in the assay solution (Supplementary 2), the use of
Eq. (S7) and a conditional association constant of Cd(II)-G complexes, β′2
2
(Supplementary 1), provides the PC production rate at a constant level of
2
free Cd(II), which can be expressed as:
2
V m′ ax½Gꢀ
+
t
v =
ð2Þ
KmB
β ½CdðIIÞꢀ
Â
2
t
KmA Gꢀ + ½Gꢀ
′
t
2
A further confirmation of the hypothesis was made using a PCS
activity assay system at a constant free Cd(II) level. For this purpose,
where V m′ ax is nominal, because the fraction of Cd(II)-bound rAtPCS1 is
unknown. Direct curve fitting of the PCS activity data to Eq. (2) was
unsuccessful probably due to the lower contribution of KmB to the reaction
rates at higher GSH concentrations, as revealed by linear relation at GSH
concentrations above 25 mM in the Hanes plot (Fig. 4B). For these reasons,
KmA and V ′m ax were determined using activity data at GSH concentrations
of 25–50 mM on the assumption that KmB was zero, followed by
optimization of KmB for the whole set of activity data using the determined
PCS activity was determined at various concentrations of total GSH
2
while the ratio of [Cd(II)]
t
to [G]
t
was maintained constant. As shown
in Fig. 4, PCS activity increased steadily with GSH concentration, in
contrast to the assay system with a constant total Cd(II) concentra-
tion, in which the PCS activity declined at higher GSH concentrations.
These results demonstrated that the reduced PCS activity at higher
GSH concentrations in the PCS assay system at a constant total Cd(II)

S. Ogawa et al. / Journal of Inorganic Biochemistry 105 (2011) 111–117
115
and a KE1 of (28.4± 5.1)×10 M−1. The Vmax and KE1 values estimated
from the activity determined at 10 μM total Cd(II) are fairly consistent
with those obtained at 5 μM Cd(II), although a difference was
apparent; the Vmax obtained at 10 μM total Cd(II) was 56% higher
than that at 5 μM Cd(II) and the KE1 obtained at 10 μM total Cd(II) was
44% lower than that obtained at 5 μM. In both cases, the parameter
obtained consistently simulated the PCS activity determined exper-
imentally, as shown by the solid lines (b and c) in Fig. 1 for the total Cd
(II) concentrations of 5 and 10 μM, respectively. In contrast, the PCS
activity at 1 μM total Cd(II) yielded a much higher KE1 value than that
obtained using 5 or 10 μM total Cd(II), with a correlation coefficient
less than that at 5 or 10 μM total Cd(II) (Table 1). This may be a result
of the poor data quality obtained at 1 μM total Cd(II), in which all of
the activity plots except that of the origin revealed a decreasing
profile; consequently, simulation of the activity using the estimated
9
K
K
mA and V ′m ax values. The obtained parameters, V′max =149± 9,
mA =18.0± 3.2 mM, and KmB =5.14± 1.22 μM, are consistent with the
experimentally determined activity because these parameters success-
fully simulated the PCS activity, as indicated by the solid line in Fig. 4A.
Furthermore, the Michaelis constants for GSH and Cd-G complexes are in
2
fair agreement with those reported previously [8], which supports the
suggestion that PC production proceeds by a substituted-enzyme
mechanism in which GSH donates a γ-glutamylcysteinyl group to the
rAtPCS1 molecule and then the Cd(II)-G
glutamylcysteinyl group in PC synthesis.
2
complex accepts the γ-
2
Our finding that the PCS synthesis rate followed Michaelis–
Menten-type kinetics at a constant free Cd(II) level indicated that in
2
Eq. (2) the KmB/β′
to (KmA/[G]
means that in the GSH concentration range, the Cd(II)-G
enzyme was almost fully occupied by the complex due to the
elevation of Cd(II)-G concentration (from 31 to 120 μM at GSH
concentrations from 25 to 50 mM). Nonetheless, no apparent
inhibition of PCS activity was observed, indicating that there is no
inhibitory GSH binding site on the enzyme.
2
[Cd(II)][G]
t
value becomes negligible with respect
t
+1) at GSH concentrations greater than 25 mM. This
site of the
2
V
max and KE1 did not adequately correlate with the experimentally
determined activity, as shown by the solid line (a) in Fig. 1.
max and KE1 were optimized for the PCS activity of the assay
solutions containing 50 μM and 500 μM total Cd(II). On the whole, the
parameters obtained at 50 μM total Cd(II) were consistent with those
obtained at 5 or 10 μM, although KE1 was 60% greater than that
obtained at 5 μM total Cd(II) (Table 1). On the other hand, the
optimization of the parameters at 500 μM total Cd(II) was unsuccess-
ful due to the extraordinarily high KE1 value (Table 1). The Vmax and
2
V
4
.3. Kinetic analysis of PC
2
synthesis mediated by rAtPCS1 at a constant
total Cd(II) concentration
It is apparent that Cd(II) activates rAtPCS1 by binding to a site on
the enzyme. Given the binding constant of Cd(II) to rAtPCS1, KE1, the
amount of active enzyme can be expressed by the following equation:
KE1 values obtained for PCS activity at higher levels of total Cd(II) (50
and 500 μM) yielded a lower correlation coefficient and a larger
standard error in the KE1 value than those estimated from the activity
determined at 5 or 10 μM total Cd(II). These differences may stem
from the sigmoidal profile of the activity at lower concentrations of
GSH in the presence of 50 or 500 μM total Cd(II) (Fig. 2).
½CdðIIÞ−rAtPCS1ꢀ = KE1½CdðIIÞꢀ½rAtPCS1ꢀ:
ð3Þ
Taking into account that only rAtPCS1 bound to Cd(II) has PCS
activity, the reaction rate can be derived as a one-site model, as
follows:
4.4. Inhibitory second Cd(II) binding site of rAtPCS1
As shown in Fig. 3, the PC synthesis activity reached a maximum
with respect to total Cd(II) concentration when the total GSH
concentration was kept constant, and the maxima shifted toward
higher Cd(II) concentrations with higher GSH concentrations. The
optimum total Cd(II) concentration has also been demonstrated for
AtPCS1 and PCS from soybean [7]. These findings suggest that rAtPCS1
possesses a second Cd(II) binding site with weaker affinity than the
first site, and that the binding of Cd(II) to this site induces an
inhibitory effect on PC synthesis. Given the binding constant of Cd(II)
to Cd(II)-rAtPCS1 at the second site, KE2, the concentration of enzyme
f V ½Gꢀt
1
max
v =
ꢀ
ꢁ
ð4Þ
K
mB
CdðIIÞꢀt
KmA
+
1 +
½Gꢀt
½
where f
1
represents the fraction of enzyme in an active form, which is
expressed by:
1
f1
=
ð5Þ
′
2
t
β
½Gꢀ
2
1
+
⋅
KE1 ½CdðIIÞꢀ
with two Cd(II) binding sites occupied by Cd(II) (Cd(II) -rAtPCS1) can
t
2
be expressed by the following equation:
Here, the unit of Vmax is μmol min− mg−1 protein, in which the
1
Â
Ã
2
enzyme is supposed to be 100% active. The parameters Vmax and KE1
were optimized for the GSH concentration-dependent PCS activity
shown in Fig. 1, where the KmA and KmB values obtained were
employed. As shown in Table 1, PCS activity at 5 μM total Cd(II)
CdðIIÞ −rAtPCS1 = KE1KE2½CdðIIÞꢀ ½rAtPCS1ꢀ
ð6Þ
2
Assuming that rAtPCS1 with two Cd(II) binding sites fully occupied
by Cd(II) ions completely loses its activity, the following equation of a
two-site model can be obtained:
−
1
−1
concentration yielded a Vmax of 193± 17 μmol min mg
protein
f2Vmax½Gꢀt
Table 1
v =
ꢀ
ꢁ
ð7Þ
KmB
Estimation of the affinity constants of Cd(II) to rAtPCS1 determined by enzyme activity
as a function of the total GSH concentration using a one-site model.
KmA
+
1 +
½Gꢀt
a)
½CdðIIÞꢀt
Cd(II) (μM)
Range of
GSH (mM)
V
max (μmol min−
1
K )
E1/109 (M−1
R2b)
mg− protein)
1
where f
following equation:
2
represents the fraction of activated enzyme according to the
1
5
0–50
0–50
0–50
0–50
0–80
152± 22
193± 17
302± 25
255± 27
205± 16
269± 102
28.4± 5.1
15.9± 2.7
45.5± 26.0
0.681
0.926
0.938
0.870
0.771
1
5
5
0
0
00
1
f2
=
ð8Þ
0
Gꢀ2t
β
½
K
½CdðIIÞꢀ
3.45×101
8c)
1 +
2
⋅
+
E2
⋅
t
0
2
KE1 ½CdðIIÞꢀt
β
½Gꢀt
2
a)
b)
Figures represent estimated value± standard error.
represents the correlation coefficient between experimentally determined
R
The binding constants KE1 and KE2 were optimized to fit PCS
activity according to Eq. (7) as a function of total Cd(II) concentration
activity and that estimated using the one-site model.
c)
Standard error exceeded the limit that can be processed in the system.

116
S. Ogawa et al. / Journal of Inorganic Biochemistry 105 (2011) 111–117
Table 2
between determinations. Among the Vmax and KE1 values obtained by
applying the one-site model, the parameters estimated from the PCS
activity determined at 5 and 10 μM total Cd(II) are likely reasonable
estimates (Table 1). The parameters obtained at 1 μM Cd(II) seemed
to be erroneous due to poor data quality. The parameters obtained at
50 and 500 μM Cd(II) also seemed to be incomplete, because the plots
exhibited sigmoidal profiles at lower GSH concentrations. The
parameters obtained using the two-site model are likely to be
consistent, although the KE1 at 500 μM total Cd(II) represented
approximately 20% of that obtained from the data sets at 5 or
Estimation of the affinity constants of Cd(II) to rAtPCS1 determined under conditions
with various concentrations of total Cd(II) with a constant total GSH concentration.a)
max (μmol min−
1
K ) K )
E1/109 (M−1 E2/109 (M−1 R2b)
GSH
Range of
V
mM) Cd(II) (μM) mg− protein)
1
(
5
2
0–100
0–500
244± 17
194± 14
23.4± 9.8
25.3± 8.6
0.557± 0.081
0.403± 0.085
0.986
0.968
0
a)
b)
Figures represent estimated value± standard error.
represents the correlation coefficient between experimentally determined
R
activity and that estimated using the two-site model.
2
0 mM total GSH with varying total Cd(II) concentration (Table 2
versus Table 3). Averaging these values amounted to Vmax =301±
−
1
−1
10
−1
4
K
8 μmol min
mg
protein, KE1 =(1.86± 0.61)×10
M
, and
8
−1
(
Table 2). Simulations of PCS activity using the Vmax, KE1, and KE2
E2 =(7.11± 2.83)×10 M , where the parameters obtained using
values thus obtained for PCS activity at 5 and 20 mM GSH were in
accordance with the experimentally obtained PCS activity in both
cases, as shown by the solid lines in Fig. 3A and B. Furthermore, the
parameters estimated from PCS activity at 5 mM GSH were consistent
with those obtained at 20 mM GSH: the Vmax, KE1, and KE2 estimated
from the activity determined at 5 mM GSH were 125%, 92.5%, and
the one-site model with PCS activity at 1, 50, and 500 μM total Cd(II)
were omitted.
The KE2 value may be greater than the value presented in this
study, because an assumption was made in the optimization that PCS
activity is completely lost when the second Cd(II) binding site of
rAtPCS1 is occupied by Cd(II). However, the observation that the PCS
activity of rAtPCS1 determined at 500 μM Cd(II) represented only 8%
of that at 4 μM Cd(II) (Fig. 3A) indicates that the enzyme bound by
two Cd(II) ions seemed to have very limited PCS activity. This was
supported by the optimization practice, in which consistent para-
meters can be obtained only when the PCS activity of the enzyme
bound by two Cd(II) ions supposedly had less than 3% enzyme
activity.
1
38% of those at 20 mM GSH, respectively. These results demonstrat-
ed unequivocally that, in addition to the first Cd(II) binding site that is
essential for activation of the enzyme, rAtPCS1 possesses a second Cd
(
II) binding site that is responsible for inactivation of the enzyme. A
possible alternative interpretation for the reduced PCS activity in the
presence of higher Cd(II) concentrations is partial denaturation of the
enzyme. However, this contribution seemed to be excluded as the
level of free Cd(II) was still low even at the highest total Cd(II)
concentration in Fig. 3A, which was calculated to be 49 nM.
The optimization of PC activity at a constant free Cd(II)
concentration gave a nominal Vmax value (V′max). Using a calculated
−
10
A sigmoidal increase in activity was observed at GSH concentra-
tions of 0–20 mM for the assay containing 50 and 500 μM total Cd(II)
free Cd(II) level of 1.22×10
rAtPCS1 yielded fractional concentrations of rAtPCS1, Cd(II)-rAtPCS1,
and Cd(II) -rAtPCS1 of 28.8%, 65.5%, and 5.7%, respectively. From
M, the association constant of Cd(II) to
(
Fig. 2). This is particularly true for the PCS assay performed in the
presence of 500 μM total Cd(II). It is likely that an increase in free Cd
II) level at lower GSH concentrations is responsible for this
phenomenon, because under such conditions, high levels of free Cd
II) may be present in the assay solution, and Cd(II) may occupy the
2
−
1
− 1
these values, Vmax was calculated to be 227 μmol min
mg
(
protein, in reasonable agreement with the averaged value.
(
second binding site of rAtPCS1, suppressing the activity. This type of
inhibition of PCS activity is anticipated to be more severe at lower
concentrations of total GSH. For these situations, Eq. (7) was
employed again to follow the PCS activity determined as a function
of total GSH concentration. Simulations of PCS activity using the
optimized parameters, which are shown in Table 3, were consistent
with the experimentally determined PCS activity in the presence of 5
and 20 mM GSH, as shown by the solid lines in Fig. 3A and B. An
improvement in the correlation coefficient between simulated and
experimentally determined PCS activity was apparent when the two-
site model was employed. This also supports the suggestion that
rAtPCS1 possesses two Cd(II) binding sites per molecule.
4.6. Comparison to other models
A kinetic analysis of PC synthesis catalyzed by AtPCS1-FLAG
indicated a substituted-enzyme mechanism where GSH and Cd(II)-G
play roles as co-substrates, with the enzyme already in an active form
without Cd(II) binding [8]. Although supportive of a substituted-
enzyme mechanism, our results demonstrated that the enzyme
requires Cd(II) binding for activation.
2
According to the model proposed by Vatamaniuk et al., the enzyme
is active without bound Cd(II), as indicated by the much lower level of
estimated free Cd(II) compared to the dissociation constant of Cd(II)
to AtPCS1-FLAG [8]. In their study, equilibrium dialysis was performed
to estimate the dissociation constant of Cd(II) to AtPCS1-FLAG, which
demonstrated that the protein possessed seven Cd(II) binding sites
with a dissociation constant of 5.6 μM. The free Cd(II) level was
4
.5. Affinity constants of rAtPCS1 to Cd(II) ions
−
13
calculated to be 6.638×10
M in the reaction medium containing
It has been demonstrated unequivocally that rAtPCS1 possesses
two Cd(II) binding sites, although the affinity constants varied slightly
total Cd(II) and GSH at concentrations of 25 μM and 3.3 mM,
respectively, buffered at pH 8.0, using the computer program SOLCON.
However, determination of the dissociation constant was performed
by fitting a single hyperbolic curve as a function of Cd(II)
concentration, although the protein apparently had seven Cd(II)
binding sites [8]. In this context, this value represents an overall
average dissociation constant and one or more of the sites may exhibit
much lower dissociation constants. Peptide scanning analyses for
TaPCS1 and SpPCS indicated that Cd(II) can still bind these PCSs in the
presence of 1 mM GSH [9]. Furthermore, the stability constants
employed by Vatamaniuk et al. [8] were so high that the estimated
free Cd(II) level was three orders of magnitude less than those
calculated with the constants from other sources [17]. From the
conditional stability constants obtained in the current study, the free
Table 3
Estimation of the affinity constants of Cd(II) to rAtPCS1 determined by the enzyme
a)
activity as a function of the total GSH concentration using a two-site model.
max (μmol min−1
E1/109 (M−1
E2/109 (M−1
)
R2b)
Cd(II) Range of
V
K
)
K
−
1
(
μM) GSH (mM) mg
protein)
5
5
0
00
0–50
0–80
399± 62
472± 94
14.2± 4.6
4.55± 1.88
0.961± 0.442
0.924± 0.333
0.971
0.984
a)
b)
Figures represent estimated value± standard error.
represents the correlation coefficient between experimentally determined
R
activity and that estimated using the two-site model.

S. Ogawa et al. / Journal of Inorganic Biochemistry 105 (2011) 111–117
117
Cd(II) level was demonstrated to be 5.6×10− M. Therefore, it can be
9
Appendix A. Supplementary data
concluded that Cd(II) binds rAtPCS1 at two sites of the enzyme.
Supplementary data to this article can be found online at
doi:10.1016/j.jinorgbio.2010.09.011.
4
.7. Potential physiological relevance of the Cd(II) binding sites of
rAtPCS1
As suggested previously [5], the first Cd(II) binding site on rAtPCS1
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reach a level that would allow the ion to bind to the second site.
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Abbreviations
AtPCS1 Arabidopsis thaliana PCS1
AtPCS1-FLAG AtPCS1 with a C-terminal FLAG tag
BCS
bathocuproinedisulfonate
[
[
Cd(II)-G
2
1:2 Cd(II) GSH complex
[
Cd(II)]
CHES
desglycyl PC
t
total concentration of Cd(II)
N-cyclohexyl-2-aminoethanesulfonic acid
4
2
2
2
PC devoid of the C-terminal glycine;
[
G
GSH with various ionized states
total concentration of G
[G]
t
,
GSH
glutathione
HEPES
2-[4-(2-hydroxyl)-1-piperazinyl]ethanesulfonic acid
[
19] L.A. del Río, L.M. Sandalio, F.J. Corpas, J.M. Palma, J.B. Barroso, Plant Physiol. 141
2006) 330–335.
rAtPCS1 recombinant AtPCS1
(
TFA
PC
trifluoroacetic acid
phytochelatin
PC
PCS
n
phytochelatin with n γ-Glu-Cys units
phytochelatin synthase