Role of substrate reactivity in the glutathione peroxidase (gpx) activity of selenocystine

Article abstract of DOI:10.1246/bcsj.20090348

Selenocystine (CysSeSeCys), a diselenide, exhibits glutathione peroxidase (GPx) activity, where it catalyses the reduction of hydroperoxides using a thiol co-factor. To understand the relative reactivity of the two substrates, enzymekinetic parameters, i.e., the turnover number (k_cat) and the relative reactivity parameters toward thiol (φ_G) and hydroperoxide (H), were determined by applying Dalziel kinetics for a bi-substrate model in the presence of hydrogen peroxide (H₂O ₂), t-butyl hydroperoxide, or α-cumyl hydroperoxide and glutathione or dithiothreitol (DTT_red). The intermediates formed during the reaction of CysSeSeCys with H₂O₂ and DTT _red were characterized by ⁷⁷SeNMR spectroscopy. Ab initio calculation at HF/6-31G(d) indicated that the reactions with H2O2 are exothermic, while those with DTTred are endothermic. Based on these studies, the GPx activity of CysSeSeCys is likely to be initiated by the reaction with hydroperoxide and in the catalytic cycle, the reaction with thiol is the rate-determining step.

Full text of DOI:10.1246/bcsj.20090348

© 2010 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 83, No. 6, 703–708 (2010)
703
Role of Substrate Reactivity in the Glutathione Peroxidase (GPx)
Activity of Selenocystine
Beena G. Singh,1 Partha P. Bag,1 Fumio Kumakura,2 Michio Iwaoka,*2 and K. Indira. Priyadarsini*1
1Radiation and Photochemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400 085, India
2Department of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka 259-1292
Received December 28, 2009; E-mail: miwaoka@tokai.ac.jp
Selenocystine (CysSeSeCys), a diselenide, exhibits glutathione peroxidase (GPx) activity, where it catalyses the
reduction of hydroperoxides using a thiol co-factor. To understand the relative reactivity of the two substrates, enzyme
kinetic parameters, i.e., the turnover number (kcat) and the relative reactivity parameters toward thiol (ºG) and
hydroperoxide (ºH), were determined by applying Dalziel kinetics for a bi-substrate model in the presence of hydrogen
peroxide (H2O2), t-butyl hydroperoxide, or ¡-cumyl hydroperoxide and glutathione or dithiothreitol (DTTred). The
intermediates formed during the reaction of CysSeSeCys with H2O2 and DTTred were characterized by 77Se NMR
spectroscopy. Ab initio calculation at HF/6-31G(d) indicated that the reactions with H2O2 are exothermic, while those
with DTTred are endothermic. Based on these studies, the GPx activity of CysSeSeCys is likely to be initiated by the
reaction with hydroperoxide and in the catalytic cycle, the reaction with thiol is the rate-determining step.
Selenium, an essential micronutrient is present in many
NH2
NH2
NH2
CO2H
redox active proteins mainly in the form of selenocysteine. To
date around 25 selenoproteins are known in humans, out of
which glutathione peroxidase (GPx) is one of the most studied
selenoenzymes.1 GPx being an oxidoreductase enzyme reduces
various hydroperoxides at the expense of thiol and therefore
acts as an endogenous antioxidant enzyme. The enzyme has a
tetrameric structure, in which each subunit contains a seleno-
cysteine residue.2 The mechanism of GPx activity in different
protein variants is controlled by the redox property of
selenocysteine present in the enzyme.3
SeH
Se Se
HO2C
HO2C
CysSeSeCys
CysSeH
NH2
NH2
O
Se
Se
HO2C
OH
HO2C
OH
CysSeOH
CysSeO2H
There is growing interest in developing low molecular
weight selenium compounds that act as GPx mimics. Several
aliphatic and aromatic organoselenium compounds, e.g.,
ebselen, benzoselenazolinones, selenenamides, selenoethers,
various diselenides, selenocysteine derivatives, and peptides,4-7
have been synthesized and evaluated for GPx activity. The
studies so far have focused on the effects of the structure,
substitution, and electrophilicity of the selenium atom. How-
ever, most of the experiments using selenium compounds as
GPx mimics were performed in organic solvents, which may
not act as a good model for the compounds with ionizable
functional groups.4 Yasuda et al. have studied the GPx-like
enzyme kinetics of selenocystine (CysSeSeCys) (Chart 1)
applying Lineweaver-Burk (L-B) plots.8 However, there is
no comprehensive report quantitatively correlating the enzyme
kinetics parameters with the energetics of the intermediate
steps.
Chart 1. Active intermediates that would be involved in the
GPx catalytic cycle of selenocystine (CysSeSeCys). The
amino, carboxylic, selenol (-SeH), and seleninic acid
(-SeO2H) groups should be ionized in neutral water.
both oxidation and reduction, therefore exhibiting better GPx
activity.9 However, it was not clear which one of the two initial
processes is crucial in the catalytic cycle. In order to resolve
this, in the present investigation roles of individual substrates
involved in the GPx catalytic cycle of CysSeSeCys have been
monitored by following enzyme kinetics, 77Se NMR spec-
troscopy, and quantum chemical calculations.
Results and Discussion
Kinetic Analysis. A diselenide like CysSeSeCys enters the
GPx catalytic cycle by two pathways, either by reduction or
by oxidation.4 In the reduction pathway, a diselenide reacts
with thiol (RSH) to form selenol (CysSeH), which in turn
reduces hydroperoxide through the formation of selenenic acid
(CysSeOH). This CysSeOH is converted back to CysSeH,
through the intermediacy of selenenyl sulfide (CysSeSR). In
the oxidation pathway, CysSeSeCys directly reacts with hydro-
Recently our group has initiated work on understanding the
role of redox processes in the antioxidant GPx activity of
diselenides like CysSeSeCys and its derivatives having either
an amino or carboxylic group, both in solutions and in vitro
models.9-11 The results indicated that CysSeSeCys containing
both amino and carboxylic functional groups could undergo
Published on the web May 28, 2010; doi:10.1246/bcsj.20090348

704
Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
GPx Activity of Selenocystine
peroxide to form CysSeOH and seleninic acid (CysSeO2H),12
and CysSeO2H should react with three molecules of RSH to
produce CysSeSR and RSSR.13 The preference between the
two pathways would depend upon the reactivity of the
diselenide with the thiol or the hydroperoxide. These reactions
are represented by eqs 1-6.
NH2
O
H
HO
N
SH
HO2C
N
CO2H
H
SH
HO
O
SH
DTTred
GSH
Chart 2. Thiol substrates investigated as a reductant in the
GPx catalytic reaction. The amino and carboxylic groups
should be ionized in neutral water.
1=2CysSeSeCys þ H2O2
k1
ꢀ! 1=2CysSeOH þ 1=2CysSeO2H þ 1=2H2O
ð1Þ
ð2Þ
ð3Þ
ð4Þ
ð5Þ
k2
1=2CysSeSeCys þ RSH ꢀ! CysSeH þ 1=2RSSR
4.5
k3
6
36
(b)
CysSeH þ H2O2 ꢀ! CysSeOH þ H2O
(a)
k4
3.0
4
CysSeOH þ RSH ꢀ! CysSeSR þ H2O
30
k5
1.5
CysSeSR þ RSH ꢀ! CysSeH þ RSSR
2
24
k6
0
300 600 900 1200
1/[GSH], M-1
0
300 600 900
CysSeO2H þ 3RSH ꢀ! CysSeSR þ RSSR þ 2H2O ð6Þ
1/[GSH], M-1
18
12
6
Enzyme kinetics is a useful tool for understanding the role
of these pathways in the actual catalytic cycle of CysSeSeCys.
So far researchers have been using L-B plots, which are
suitable for single substrate models.14 However as the GPx
catalytic activity involves two substrates, the kinetic data
need to be treated with the Dalziel equation, as applied for
several multi-substrate models.15,16 Accordingly, in the present
study, the enzyme kinetic parameters were estimated using
eq 7.
1 mM GSH
1.5 mM GSH
2 mM GSH
3 mM GSH
4 mM GSH
0
0
5000
10000
15000
1/[H2O2], M-1
20000
25000
30000
½Etꢁ
ºG
ºH
ºGH
Figure 1. Representative double-reciprocal plots according
to Dalziel equation (eq 7) for CysSeSeCys-catalyzed
reduction of H2O2 in the presence of different concen-
trations of GSH. Insets (a) and (b) show secondary Dalziel
plots in accordance with eqs 8 and 9, respectively.
¼ º0 þ
þ
þ
ð7Þ
¹
½Thiolꢁ ½H2O2ꢁ ½Thiolꢁ½H2O2ꢁ
Here [Et] is the total enzyme concentration. CysSeSeCys being
a diselenide produces two reactive species either during
oxidation or reduction that can independently participate in
the GPx-like cycle (eqs 1 and 2). Therefore, in this case the
total enzyme concentration is twice the concentration of
CysSeSeCys. In eq 7, ºG is the reciprocal of the total reactivity
of the enzyme and its intermediates with thiol [ºG = 1/kG =
1/(k2 + k4 + k5 + k6)], ºH is the reciprocal of the total
reactivity of the enzyme and its intermediates with hydro-
peroxide [ºH = 1/kH = 1/(k1 + k3)], and ºGH is the parameter
related to the rate of formation of the ternary complex. The
values kG and kH are the apparent rate constants for the overall
reactions involving thiol and hydroperoxide, respectively. A
nonzero value of ºGH indicates formation of a ternary complex
between CysSeSeCys and both the substrates. º0 is equal to the
reciprocal of the turnover number which corresponds to the
maximum catalytic rate at unit enzyme concentration (º0 =
1/kcat). To estimate these parameters, a series of experiments
were performed and the initial reduction rate of hydrogen
peroxide (H2O2) (¹) was measured in the presence of
CysSeSeCys (0.1 mM) at a fixed initial concentration of thiol
[either glutathione (GSH) or dithiothreitol (DTTred) (Chart 2)]
and different concentrations of H2O2 (45-700 ¯M). A linear
plot was obtained for the variation of [Et]/¹ as a function of the
reciprocal concentration of H2O2. The slope and intercept of the
plot are represented by eqs 8 and 9.
ºG
Intercept ¼ º0 þ
½Thiolꢁ
ð9Þ
Similar experiments were repeated with different concen-
trations of thiol from 1.0 to 4.0 mM, at a variable concentration
of H2O2 (45-700 ¯M) and a fixed concentration of CysSeSeCys
(0.1 mM), to obtain different slopes and intercept values. The
primary plots from all these individual studies did not appear to
be parallel (Figure 1 and Figure S1), further suggesting the
possibility of the ternary complex formation.15,16 Therefore, the
slope values obtained from these primary plots were plotted as
a function of the reciprocal thiol concentration (i.e., [GSH] or
2[DTTred]) according to eq 8, and from the slope and intercept
of this secondary plot, ºGH and ºH values, as listed in Table 1,
have been derived (Inset (a) of Figure 1). Similarly, the
different intercept values obtained from the primary plots at
different concentrations of thiol, when plotted against the
reciprocal concentration of thiol, gave a secondary plot (Inset
(b) of Figure 1). The slope and intercept of this secondary plot,
according to eq 9, correspond to ºG and º0, respectively, as
listed in Table 1. The ºG values for the two thiol substrates
indicate that the reactivity of CysSeSeCys and its intermediates
is higher with DTTred than that with GSH. Comparison of the
ºH values with ºG values (Table 1) confirms the higher
reactivity of CysSeSeCys and the intermediates with H2O2 than
that with either of the thiols.
ºGH
Slope ¼ ºH þ
ð8Þ
½Thiolꢁ

B. G. Singh et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
705
Table 1. Kinetic Coefficients and Apparent Rate Constants of CysSeSeCys for the Reduction of Hydroperoxides by Thiola)
¹4
¹2
º0
/min
kcat
/min
ºH © 105
/M min
kH © 10
ºG © 103
/M min
kG © 10
ºGH © 107
/M2 min
Peroxides
Thiols
/M¹1 min
/M¹1 min
¹1
¹1
¹1
H2O2
GSH
1.23
(«0.12)
0.61
(«0.05)
0.88
0.81
(«0.07)
1.63
(«0.12)
1.13
1.9
(«0.9)
1.8
(«0.2)
9.5
5.2
(«0.4)
5.5
(«0.5)
1.0
4.3
(«0.4)
1.9
(«0.2)
4.1
2.3
(«0.2)
5.3
(«0.4)
2.4
4.4
(«0.5)
1.1
(«0.1)
6.1
DTTred
GSH
CumOOH
(«0.09)
1.64
(«0.1)
0.61
(«1.2)
20
(«0.1)
0.50
(«0.4)
7.2
(«0.1)
1.3
(«0.5)
0.26
t-BuOOH
GSH
(«0.15)
(«0.05)
(«1.8)
(«0.04)
(«0.7)
(«0.12)
(«0.03)
a) The kinetic parameters listed were obtained by Dalziel analysis using eqs 7-9. See the text for details.
Similarly the reactivity of other hydroperoxides, ¡-cumyl
hydroperoxide (CumOOH) (Figure S2) and t-butyl hydroper-
oxide (t-BuOOH) (Figure S3), toward CysSeSeCys was esti-
mated by using GSH as the thiol substrate. The º0, ºH, ºG,
and ºGH obtained from these studies are listed in Table 1. As
observed above, similar non-parallel primary plots were
observed, confirming formation of a ternary complex. Com-
parison of the ºH values for the three hydroperoxides suggests
that the reactivity of CysSeSeCys is the highest with H2O2
followed by those with CumOOH and t-BuOOH. It can also be
confirmed that the reactions of CysSeSeCys and its catalytic
intermediates with thiol are slower than those with hydro-
peroxide.
The reaction of hydroperoxide with CysSeSeCys is initiated
by a two-electron transfer process.17 Therefore, the oxidation of
CysSeSeCys would be controlled by redox potentials of the
hydroperoxides. Two-electron reduction potential (E°) values
for H2O2, CumOOH, and t-BuOOH are 1.76, 1.198, and
1.078 V vs. NHE, respectively.18 This shows that among the
hydroperoxides, H2O2 is the strongest oxidizing agent and
therefore facilitates the easier electron transfer from CysSeSe-
Cys to H2O2 leading to a higher kH value. Similarly, the
reaction with thiols should also be redox controlled as DTTox/
DTTred has a more negative reduction potential (¹0.332 V) than
GSSG/GSH (¹0.240 V).19,20 Other factors such as structural
differences in thiols and hydroperoxides may also significantly
control the GPx activities.21 The large kcat value in the presence
of DTTred (1.63 min¹1) would be in part due to the dithiol
structure which facilitates the conversion from CysSeSR to
CysSeH (eq 5) because the reaction becomes a unimolecular
process. The small kG value in the presence of t-BuOOH
(1.3 © 102 M¹1 min¹1) may be due to indirect effects of the
sterically bulky t-Bu group that are not known at this moment.
77Se NMR Analysis. After estimation of the enzymatic
parameters for CysSeSeCys, characterization of the catalytic
intermediates was attempted by 77Se NMR spectroscopy. The
study was restricted to DTTred as the thiol substrate and
H2O2 as the hydroperoxide substrate. The reason for selection
of these two specific substrates is that unlike other substrates,
DTTred and H2O2 are simpler compounds and exhibit minimum
interference in the NMR spectra. Since CysSeSeCys has
limited solubility at pH 7, the NMR experiments were carried
out at pH 10, where the selenol (RSeH), seleninic acid
(RSeO2H), and thiol (RSH) functional groups should be
ionized.
In the first step, CysSeSeCys was treated with a stoichio-
metric amount (1:1) of DTTred. The 77Se NMR signal of
CysSeSeCys at 272 ppm disappeared completely within twenty
minutes and a new peak was observed at ¹242 ppm, indicating
¹
quantitative conversion of CysSeSeCys to selenolate (CysSe )
(Figure S4) as also observed earlier by Tan et al.22 This
complete and quantitative conversion can be rationalized by
redox potentials of the individual couples for the equilibrium
shown in eq 10.
CysSeSeCys þ DTTred ꢀ 2CysSeꢀ þ DTTox þ 2Hþ ð10Þ
The
reduction
potential
values
for
SeCys
ꢀ;Hþ
ðECysSeSeCys=2CysSe
Þ and DTT (EDTTox/DTTred) at pH 7 are
¹0.383 and ¹0.332 V vs. NHE, respectively,19 indicating that
the reaction should not be spontaneous. However, the reduction
potential of DTT would decrease to ¹0.510 Vat pH 10 because
the pKa values for the thiol groups are 9.26 and 10.34. This
favorable potential change would be responsible for quantita-
¹
tive conversion of CysSeSeCys to CysSe under the NMR
experiment conditions. As expected, treating CysSeSeCys with
10 molar equivalents of DTTred at pH 1 did not show any
CysSeH formation.
In the second step, an equimolar amount of H2O2 was added
¹
to the above CysSe solution. Two signals were observed at
272 and 1183 ppm in the 77Se NMR spectrum, which could be
¹
assigned to CysSeSeCys and CysSeO2 , respectively: the pKa
values for areneseleninic acids are 4-5.23 The ratio of the
yields was 2:1 according to the integrals of the 1H NMR
¹
absorptions. The formation of the seleninate anion (CysSeO2
)
is expected to be through the intermediacy of CysSeOH,
which being highly reactive would be trapped either by
¹
CysSe to yield CysSeSeCys24 or by excess H2O2 to yield
CysSeO2 . The assignment of 77Se NMR peak at 1183 ppm to
¹
¹
CysSeO2 is based on an earlier observation by House et al. for
BocNHCH2CH2SeO2H at 1187 ppm at pH 8.0.25
In the third step, CysSeSeCys was treated with a stoichio-
metric amount (1:1) of H2O2 and the reaction was monitored
for 24 h. The 77Se NMR signal at 272 ppm decreased and a
¹
new peak, corresponding to CysSeO2 , appeared at 1183 ppm
(Figure 2). The reaction was completed in three hours without
formation of any other products. Under these conditions, the
¹
yield of CysSeO2 was estimated to be 33% based on the
1H NMR spectral integration data. The result clearly shows that
the oxidation of CysSeOH with H2O2 is much faster than that
of CysSeSeCys (eq 1). When CysSeSeCys was treated with 3

706
Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
GPx Activity of Selenocystine
CysSeSeCys
2.309ÅSe
Se
CysSeO2H
Se
2.586Å
N
1.971Å
2.172Å
1.969Å
102.0°
102.2°
(a)
O
1.975Å
O
N
1.777Å
N
O
O
O
O
(b)
(c)
CysSeSeCys
CysSe-
EHF = -2718.99602 au
CysSeSCys
EHF = -5437.79376 au
N
O
2.027Å
N
O
2.315Å
1.748Å
99.0°
O
97.6°
2.180Å
1.795Å
1.671Å
1.949Å
O
Figure 2. A series of 77Se NMR spectra obtained in D2O
containing NaOH (pH 10) for the reaction mixtures of (a)
CysSeSeCys and H2O2 (1:1), (b) CysSeSeCys and H2O2
(1:3), and (c) CysSeSeCys and H2O2 (1:3), then L-CysSH
(6 equiv).
Se
1.651Å
O
Se
100.1°
1.924Å
O
O
-
CysSeOH
CysSeO2
EHF = -2794.30457 au
EHF = -2868.64993 au
¹
equivalents of H2O2, the yield of CysSeO2 increased up to
Figure 3. Global energy minimum structures of CysSeSe-
¹
88% without formation of any other by-products. CysSeO2
¹
¹
Cys, CysSe , CysSeOH, and CysSeO2 obtained in water
at HF/CPCM/6-31G(d). Pertinent atomic distances and
bond angles are shown. The torsion angle of CSeSeC in
CysSeSeCys was ¹100.5°.
was reduced back to CysSeSeCys by the subsequent treatment
with DTTred probably through eq 6.13 However, we could not
observe formation of the selenenyl sulfide (CysSeSR) inter-
mediate in the reaction probably because even if it is produced,
it may be rapidly converted to cyclic DTTox and CysSe , which
in turn would react with CysSeO2 to produce CysSeSeCys.
In another experiment, when a monothiol like L-cysteine
(L-CysSH, 3 equivalents), was added to the solution, a
signal was however observed at 349 ppm in the 77Se NMR
spectrum (Figure 2), indicating formation of a selenenyl sulfide
intermediate (CysSeSCys). But no signal corresponding to
CysSeOH was observed in any of these NMR experiments.
Ab Initio Calculation. Since CysSeSeCys has ionizable
functional groups, the zwitterionic form was employed in the
ab initio calculations at HF/6-31G(d). The stable structures
¹
¹
in our model. The molecular environment around the seleno-
cysteine residue would change the conformation, thereby
modifying the antioxidant activity.27
Having obtained the HF energies (EHF) for optimized
¹
¹
structures of CysSeSeCys, CysSe , CysSeOH, CysSeO2
,
DTTred, and DTTox in water, the reaction energies (¦EHF) for
the most likely steps involved in the GPx cycle have been
calculated as shown in Table 2. The results suggest that the
reaction of CysSeSeCys with H2O2 is exothermic, while that
¹
with DTTred is endothermic. Similarly, the reaction of CysSe
¹
¹
for CysSeSeCys, CysSe , CysSeOH, CysSeO2 , DTTred, and
DTTox were located in water by exhaustive conformer search,
in which all possible dihedral angles were systematically
changed and the geometry of each structure was optimized
with the PCM model, which is frequently employed for
GPx models.24,26,27 The global energy minimum structures of
CysSeSeCys, CysSe , CysSeOH, and CysSeO2 obtained in
water are shown in Figure 3. The global energy minimum
structure of DTTred was in the extended form, and cyclic DTTox
possessed two hydroxy groups in the equatorial directions in
water (Figure S5).
with H2O2 is exothermic, while the reaction of CysSeOH with
DTTred is endothermic. This indicates that in the catalytic
reaction of CysSeSeCys in water, the reaction with hydro-
peroxide should proceed faster than that with thiol. The results
are consistent with the calculation by Bayse,30 in which three
fundamental reactions of the GPx catalytic cycle, i.e., eqs 3-5,
were followed by application of an explicit water solvent
model for a simple GPx model, benzeneselenol (PhSeH), to
¹
¹
reveal that oxidation of the selenol to the selenenic acid with
¹1
H2O2 is exothermic (¦H µ ¹60 kcal mol¹1) (1 kcal mol
=
4.184 kJ mol¹1) while reduction of the selenenic acid to
the selenol with thiol is much less exothermic (¦H µ ¹10
kcal mol¹1).
The structures shown in Figure 3 have reasonable bond
parameters around the Se atom compared with the structure of
GPx enzyme determined by X-ray analysis28 as well as those
calculated for several GPx mimics.24,26,27,29-32 However, the
torsion angle ¸(SeC¢C¡C) was in discrepancy with the X-ray
structure: Most structures shown in Figure 3 have ¸ = ca. ¹60°
Further, the global energy minimum structure of CysSeSe-
Cys obtained from quantum chemical calculation does not
show any non-bonding interaction between Se and the het-
eroatoms like O or N. Our observation is contrary to that
reported by Yasuda et al. that the GPx-like activity of
CysSeSeCys is initiated by reaction with thiol.8 Support for
our observation comes from a recent report by Mugesh
et al.,14,33 where it has been reported that in aromatic
diselenides nonbonding interactions assist in the reduction of
¹
except for CysSeO2 and the right half of the CysSeSeCys
model (¸ = ca. 180°), while at the active site of GPx the
selenocysteine residue has ¸ = ca. 60°.28 Nevertheless, the
difference in the selenocysteine structure should be due to
truncation of the other amino acid residues from the active site

B. G. Singh et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
707
Table 2. Energy Changes (¦EHF) during the GPx-Like Catalytic Cycle of CysSeSeCys in the
a)
Reaction of H2O2 with DTTred
¹1
Reactions
¦EHF/kcal mol
¹
CysSeSeCys + DTTred + 2H2O ¼ 2CysSe + DTTox + 2H3O+
94.5
¹13.8
¹88.6
25.1
¹
CysSeSeCys + 2H2O2 ¼ CysSeOH + CysSeO2 + H3O+
CysSe + H2O2 + H3O+ ¼ CysSeOH + 2H2O
¹
CysSeOH + DTTred ¼ CysSe + DTTox + H3O+
¹
a) Calculated at HF/CPCM/6-31G(d) in water. The global energy minimum structure was
employed for each species.
2H2O2
H2O
k1
CysSeSeCys
CysSeO2H
H2O
H2O2
Thermodynamically
not feasible
3GSH
[CysSeOH]
GSH
H2O
k6
2GSH
k2
H2O
GSSG
+ 2H2O
k4
k3
H2O2
GPx cycle
GSSG
CysSeH
CysSeSG
k5
GSSG
GSH
Scheme 1. A plausible catalytic cycle for the GPx-like activity of CysSeSeCys.
diselenides with thiol, while compounds without such inter-
actions react preferably with hydroperoxide rather than thiol.
peroxide were estimated by iodometric titration.34 Solutions
were prepared using water from a nanopure system. The purity
of CysSeSeCys was verified by 1H and 77Se{1H} NMR spectra
recorded on a Bruker AV500 NMR spectrometer operating
at 500 and 95.43 MHz, respectively. D2O (99.8 atom %D)
containing a small portion of NaOH (pH 10) was employed as
the solvent. Chemical shifts ¤ are given in ppm as the shifts
from CHCl3 (for 1H) and diphenyl diselenide (for 77Se) as
external standards. For the NMR experiments the concentration
of CysSeSeCys was 15 mM at pH 10 and all experiments were
carried out under inert conditions (argon atmosphere).
Conclusion
The GPx-like catalysis of organoselenium compounds
utilizes two substrates, hydroperoxide and thiol. The present
studies confirm that the GPx-like cycle of CysSeSeCys is
initiated preferably by the reaction with hydroperoxide to form
¹
CysSeOH and CysSeO2 , rather than the reaction with thiol to
¹
¹
form CysSe . CysSeOH and CysSeO2 subsequently undergo
reduction with a thiol substrate to produce CysSeSG, which is
¹
converted to the active CysSe by the reaction with another
GPx Activity.
GPx activities of CysSeSeCys were
thiol molecule. In the overall GPx cycle, the reduction with
thiol being the slowest becomes the rate-determining process.
All these possible reactions are summarized in Scheme 1.
Based on these studies it is suggested that while developing
water-soluble GPx mimics, molecular design of a diselenide
should be made in such a way that it can be easily oxidized and
the resulting intermediates can undergo easier reduction.
monitored spectrophotometrically by using either NADPH-
GSSG reductase coupled assay9,10 or by the direct oxidation
35
of DTTred
.
In the former assay, the test mixture contained
NADPH, GSH, and glutathione reductase in 0.1 M potassium
salts of phosphate buffer (pH 7.4). CysSeSeCys was added to
the mixture, and the reaction was initiated by addition of
hydroperoxide. The initial concentrations of NADPH, GSH,
glutathione reductase, CysSeSeCys, and the hydroperoxide
were 0.34 mM, 1.0-4.0 mM, 5.0 mU mL¹1, 0.1 mM, and 45-
700 ¯M, respectively. The initial reduction rate of the hydro-
peroxide (¹) was calculated from the rate of NADPH oxidation
by following the decay of absorbance due to NADPH at
340 nm. In the absence of CysSeSeCys, the decay was quite
slow. In the second assay, the peroxidase-like activity of
CysSeSeCys using DTTred as a substrate was monitored by
following the formation of DTTox at 310 nm both in the
Experimental
General.
Selenocystine, reduced nicotinamide adenine
dinucleotide phosphate tetrasodium salt (NADPH), glutathione
reductase, glutathione (GSH), reduced dithiothreitol (DTTred),
hydrogen peroxide (H2O2), t-butyl hydroperoxide (t-BuOOH),
and ¡-cumyl hydroperoxide (CumOOH) from Sigma/Aldrich
have been procured from local agents. The concentrations of
aqueous H2O2 (30% H2O2), CumOOH, and t-butyl hydro-

708
Bull. Chem. Soc. Jpn. Vol. 83, No. 6 (2010)
GPx Activity of Selenocystine
14 K. P. Bhabak, G. Mugesh, Chem. Asian J. 2009, 4, 974.
15 K. Dalziel, Acta Chem. Scand. 1957, 11, 1706.
16 H. Sztajer, B. Gamain, K.-D. Aumann, C. Slomiannyi, K.
Becker, R. Brigelius-Flohé, L. Flohé, J. Biol. Chem. 2001, 276,
7397.
absence and presence of CysSeSeCys. The initial concentra-
tions of DTTred, H2O2, and CysSeSeCys were 0.5-4.0 mM, 45-
360 ¯M, and 0.1 mM, respectively. Using ¹, the kinetic
parameters were estimated by employing Dalziel plots for a
bi-substrate model, according to a procedure given in refer-
ences.15,16
17 C. C. Winterbourn, M. B. Hampton, Free Radical Biol.
Med. 2008, 45, 549.
Ab Initio Calculation. Quantum chemical calculations
were performed by using a Gaussian 03 software package
(revision B.04).36 Stable conformers were systematically
searched by changing all the possible dihedral angles in a step
size of 120°. Geometry of each built structure was optimized at
the HF/6-31G(d) level in water by using a conductor-like
solvation model (CPCM), which is a modified form of a
polarizable continuum model (PCM).37,38 In PCM, the solvent
is modeled as a continuous static medium characterized by a
dielectric constant (¾), which is modified as a scaled conductor
boundary in CPCM. The energies were not corrected to the
zero-point energies.
18 R. Baron, A. Darchen, D. Hauchard, Electrochim. Acta
2004, 49, 4841.
19 T. Nauser, S. Dockheer, R. Kissner, W. H. Koppenol,
Biochemistry 2006, 45, 6038.
20 F. Q. Schafer, G. R. Buettner, Free Radical Biol. Med.
2001, 30, 1191.
21 M. Iwaoka, F. Kumakura, Phosphorus, Sulfur Silicon Relat.
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22 K.-S. Tan, A. P. Arnold, D. L. Rabenstein, Can. J. Chem.
1988, 66, 54.
23 J. D. McCullough, E. S. Gould, J. Am. Chem. Soc. 1949,
71, 674.
24 B. Cardey, M. Enescu, ChemPhysChem 2005, 6, 1175.
25 K. L. House, R. B. Dunlap, J. D. Odom, Z.-P. Wu, D.
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26 J. K. Pearson, R. J. Boyd, J. Phys. Chem. A 2006, 110,
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27 B. Cardey, M. Enescu, J. Phys. Chem. A 2007, 111, 673.
28 O. Epp, R. Ladenstein, A. Wendel, Eur. J. Biochem. 1983,
133, 51.
This work was carried out under DST-JSPS sponsored
collaborative program (DST/INT/JAP/P-45/08). BS, PPB,
and KIP would like to acknowledge the support and encourage-
ment from Drs. T. Mukherjee, S. K. Sarkar and V. K. Jain,
BARC.
Supporting Information
29 C. A. Bayse, J. Phys. Chem. A 2007, 111, 9070.
30 C. A. Bayse, S. Antony, J. Phys. Chem. A 2009, 113, 5780.
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Additional experimental and calculation results. This mate-
rial is available free of charge on the Web at http://www.csj.jp/
journals/bcsj/.
32 S. M. Bachrach, D. W. Demoin, M. Luk, J. V. Miller, Jr.,
J. Phys. Chem. A 2004, 108, 4040.
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