kinetic studies on the glutathione peroxidase activity of

www.elsevier.com/locate/cbpb

Comparative Biochemistry and Physiol

Kinetic studies on the glutathione peroxidase activity of

selenium-containing glutathione transferase

Huijun Yua, Junqiu Liua,*, Xiaoman Liua, Tianzhu Zangb, Guimin Luob, Jiacong Shena

aKey Laboratory for Supramolecular Structure and Materials of Ministry of Education, Jilin University,

Changchun 130012, People’s Republic of ChinabKey Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University,

Changchun 130023, People’s Republic of China

Received 26 February 2005; received in revised form 30 April 2005; accepted 3 May 2005

Available online 8 June 2005

Abstract

Selenium-containing glutathione transferase (seleno-GST) was generated by biologically incorporating selenocysteine into the active site

of glutathione transferase (GST) from a blowfly Lucilia cuprina (Diptera: Calliphoridae). Seleno-GST mimicked the antioxidant enzyme

glutathione peroxidase (GPx) and catalyzed the reduction of structurally different hydroperoxides by glutathione. Kinetic investigations

reveal a ping-pong kinetic mechanism in analogy with that of the natural GPx cycle as opposed to the sequential one of the wild type GST.

This difference of the mechanisms might result from the intrinsic chemical properties of the incorporated residue selenocysteine, and the

selenium-dependent mechanism is suggested to contribute to enhancement of the enzymatic efficiency.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Enzyme mimics; Glutathione peroxidase; Kinetics; Mechanism; Selenium; Selenium-containing glutathione transferase; Selenium-containing

glutathione transferase from Lucilia cuprina; Selenocysteine

1. Introduction

Glutathione peroxidase (GPx, EC.1.11.1.9) is a well-

known selenoenzyme that functions as an antioxidant by

catalyzing the reduction of harmful peroxides with gluta-

thione (GSH) and protecting the lipid membranes and other

cellular components against oxidative damage (Arun and

Subramanian, 1998; Gaal et al., 1995; Mahmoud and Edens,

2003; Surai et al., 1998). The selenium of the selenocysteine

(Sec) in the enzyme catalytic site undergoes a redox cycle

involving the selenol (ESeH) as the active form (Epp et al.,

1096-4959/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.cbpc.2005.05.003

Abbreviations: GPx, glutathione peroxidase; GSH, glutathione; Sec,

selenocysteine; GST, glutathione transferases; LuGST1-1, GST from

Lucilia cuprina; seleno-GST, selenium-containing GST; seleno-LuGST1-

1, selenium-containing GST from Lucilia cuprina; CuOOH, cumene

peroxide; t-BuOOH, tert-butyl peroxide; NADPH, h-nicotinamide adenine

dinucleotide phosphate, reduced form.

* Corresponding author. Tel.: +86 431 5168452; fax: +86 431 5193421.

E-mail address: [email protected] (J. Liu).

1983). The selenol is first oxidized by hydrogen peroxide or

organic peroxides to selenenic acid (ESeOH) which then

reacts with a reduced glutathione (GSH) to form selenenyl

sulfide adduct (ESeSG). Finally, the attack of a second

equivalent of reduced glutathione to ESeSG regenerates the

active form of the enzyme and simultaneously produces the

oxidized glutathione (GSSG) (Scheme 1) (Flohe et al.,

1972; Ursini et al., 1982).

Thus, in the overall catalytic cycle, 2 equivalents of

glutathione are oxidized to disulfide and water, while the

hydroperoxide is reduced to the corresponding alcohol.

Although seleninic acid (E-SeO2H) could also be formed in

the presence of high concentrations of hydroperoxide (Ren

et al., 1997), it is usually believed to lie off the main

catalytic pathway.

In recent years, there were increasing interests in

mimicking the functions of this important antioxidant

enzyme not only for elucidating catalytic mechanism but

also for potential pharmaceutical applications. Several

attempts have been made to produce GPx mimics with

ogy, Part B 141 (2005) 382 – 389

ESeO2H2GSH

ROOHESeOH

ESeSG

ESeH

GSH

GSSG

GSH

H2O

ROOHROH

Scheme 1. The catalytic mechanism of natural GPx. This figure was derived

from Flohe, 1989.

H. Yu et al. / Comparative Biochemistry and Physiology, Part B 141 (2005) 382–389 383

high efficiency such as monoclonic selenoantibody (Luo et

al., 1994), bioimprinted selenoprotein (Liu et al., 1999), and

synthetic selenium-or tellurium-containing compounds

(Mugesh and Singh, 2000; Mugesh and Du Mont, 2001;

Mugesh et al., 2001a). Especially, the redesign of existing

protein scaffolds has also been proved to be an efficient

strategy to generate novel GPx mimics (Wu and Hilvert,

1990; Boschi-Muller et al., 1998). Glutathione transferases

(GSTs, EC. 2.5.1.18) are a family of multifunctional

proteins capable of detoxifying endogenous and xenobiotic

electrophiles by addition of GSH to the electrophiles

(Armstrong, 1997; Dirr et al., 1994; Vanhaelen et al.,

2004; Zielinski and Portner, 2000). Because their enzyme

folds in the GSH-binding sites are similar to that of GPx,

GSTs have been used as excellent candidate models to gain

GPx activity by the incorporation of Sec through chemical

modification or genetic engineering (Ren et al., 2002; Jiang

et al., 2004; Yu et al., 2005). The selenium-containing GST

from blowfly Lucilia cuprina (seleno-LuGST1-1) was

generated by replacing the active site serine9 with a cysteine

and then substituting it with selenocysteine in a cysteine

auxotrophic system (Yu et al., 2005). This novel selenoen-

zyme exhibited significantly high efficiency for catalyzing

reduction of hydrogen peroxide by GSH, being comparable

with those of natural GPx, and a typical ping-pong kinetic

mechanism has been suggested for its enzymatic reaction.

Herein, we evaluated the catalytic ability of seleno-

LuGST1-1 for other structurally different hydroperoxides.

The detailed kinetic studies on various hydroperoxides

including hydrogen peroxide (H2O2), tert-butyl hydroper-

oxide (t-BuOOH), and cumene hydroperoxide (CuOOH)

have been carried out. An alternation of catalytic mecha-

nism was suggested to take place after the conversion of

Ser9 of LuGST1-1 to Sec.

2. Materials and methods

2.1. Materials

Reduced glutathione (GSH) and tert-butyl hydroperoxide

(t-BuOOH) were purchased from Merck. Cumene hydro-

peroxide (CuOOH) was purchased from Fluka. h-Nicoti-namide adenine dinucleotide phosphate reduced form

(NADPH) and glutathione reductase (type N baker’s

yeast) were obtained from Sigma-Aldrich. Glutathione

Sepharose 4B and Sephadex G-25 were purchased from

Amersham Pharmacia Biotech, Uppsala, Sweden. All other

chemicals were of the highest purity commercially

available and were used without further purification.

Spectrometric measurements were carried out with a

Shimadzu 3100 UV–vis-IR recording spectrophotometer.

Data were acquired and analyzed by using ultraviolet

spectroscopy software. The concentrations of the stock

solutions of hydroperoxide were determined by titration

with potassium permanganate.

2.2. Preparation of selenium-containing glutathione

transferase

Seleno-LuGST1-1 was expressed and purified as

described previously (Yu et al., 2005). Briefly, the plasmid

pSM3 which contained the gene of seleno-LuGST1-1 was

transformed into a cysteine auxotrophic strain

BL21cysE51. For overexpressing seleno-LuGST1-1, an

overnight culture of BL21cysE51/pSM3 grown in LB

medium was used to inoculate 1 L of M9 expression

medium, which is a modification of M9 minimal medium,

containing ampicillin, kanamycin and cysteine HCl (50 Ag/mL) to an OD600 of 0.1. When the culture had reached an

optical density of about 1, IPTG was added to a

concentration of 1 mM and after a further 10 min

chloramphenicol to a concentration of 10 Ag/mL. Five

minutes after the addition of the antibiotic the culture was

transferred into prechilled centrifuge beakers and the cells

were sedimented at 6000 rpm for 5 min. They were

washed twice in cold saline (1 L each) and resuspended in

the production medium containing 400 Ag/mL rifampicin

and 600 AM l-selenocysteine. Incubation was continued

for further 2.5 h. The cells were then harvested (10 min at

6000 rpm) and washed twice in an equal volume of 50

mM Tris–HCl buffer (pH 7.0) containing 1mM EDTA.

Then the cells were suspended in this buffer (1 mL/g cell

wet mass) containing 1 mM phenylmethanesulfonyl

fluoride and broken by sonication. The supernatant was

collected by centrifugation and applied to GSH Sepharose

4B equilibrated in 50 mM Tris–HCl buffer (pH 7.2). The

column was washed with several bed volumes of 50 mM

Tris–HCl buffer (pH 7.2), until the eluate was free of

protein as determined by A280 measurement. The seleno-

LuGST1-1 was eluted from the affinity column with 5 mM

GSH in 50 mM Tris–HCl buffer (pH 9.6), and the enzyme

fraction was dialysed and applied to Sephadex G-25

column. The purity of the enzyme was determined by

electrophoresis on a 15% SDS-PAGE. Protein concen-

tration was determined by a Bio-Rad protein assay using

BSA as a standard following the method of Bradford

(1976).

Table 1

Values of GPx activities of seleno-LuGST1-1 and other catalysts

Catalysts Substrate Activitya

(AmolImin�1IAmol�1)

Wild type LuGST1-1 H2O2 N.D.b

CuOOH 7.8T0.3

t-BuOOH 1.1T0.2

Seleno-LuGST1-1 H2O2 2980T30

H. Yu et al. / Comparative Biochemistry and Physiology, Part B 141 (2005) 382–389384

2.3. Assay of glutathione peroxidase activities

The GPx activity was assayed using the coupled assay

described by Wilson (Wilson et al., 1989). Glutathione

disulfide (GSSG) formed in the first step was reduced to

GSH by NADPH in a reaction catalyzed by glutathione

reductase. The amount of GSSG formed was followed by the

decrease in absorbance at 340 nm due to consumption of

NADPH. At the NADPH concentration used, the coupled

reaction was a zero-order reaction with respect to NADPH and

the oxidation of GSHwas the rate-limiting step. The amount of

GSH in the reaction was thus kept constant (Eqs. (1) and (2)).

2GSHþ ROOHYEnzyme

GSSGþ ROHþ H2O

R ¼ H; t � Butyl;Cumenylð1Þ

GSSGþ NADPHþ HþYGSSG reductase

2GSHþ NADPþ: ð2ÞThe reaction was carried out at 37 -C in 500 AL of 50 mM

potassium phosphate buffer, pH 7.0, containing 1mMEDTA,

1 mM sodium azide, 1 mM GSH, 1 unit of GSSG reductase,

and 10–50 nM of enzyme. The mixture was preincubated for

7 min, and then 0.25 mM NADPH solution was added and

incubated for 3 min at 37 -C. Thereafter, the reaction was

initiated by addition of 0.5 mM H2O2. The activity was

monitored as the decrease of NADPH absorption at 340 nm.

Appropriate control of the non-enzymatic reaction was

performed and subtracted from the catalyzed reaction. The

activity unit is defined as the amount of the enzyme that

catalyzes the turnover of 1 Amol of NADPH per min. The

specific activity is expressed in Amol min�1Amol�1.

2.4. Steady-state kinetics of seleno-LuGST 1-1

The assay of kinetics of seleno-LuGST1-1 for the reduction

of ROOH (R=H, t-Butyl, Cumenyl) by GSH was similar to

that of selenium-containing catalytic antibody, Se-4A4 (Ding

et al., 1998). The initial rates were measured by observing the

decrease of NADPH absorption at 340 nm at several

concentrations of one substrate while the concentration of

the second substrate was kept constant. All kinetic experi-

ments were performed in a total volume of 0.5 mL containing

50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1

unit of GSH reductase, 0.25 mM NADPH, and varying

concentrations of GSH, ROOH, and seleno-LuGST1-1. After

the enzyme was preincubated with GSH, NADPH, and GSH

reductase, the reaction was then initiated by the addition of

ROOH. Subtraction of the non-enzymatic background

absorption gave the rate of the enzyme-catalyzed reaction.

CuOOH 2071T20t-BuOOH 1021T10

Native (GPX, rabbit liver)c H2O2 5780

a Activities were determined as initial rates in 50 mM potassium

phosphate buffer (pH 7.0) at 37 -C containing 1mM GSH and 0.5 mM

hydroperoxide and corrected for the spontaneous reactions. All values

(meansTSD) are the means of at least three determinations.b N.D., no detectable GPx activity.c As previously described (Mannervik, 1985).

3. Results

3.1. The glutathione peroxidase activity of seleno-LuGST1-1

The GPx activities were determined indirectly by the

coupled assay system.

As shown in Table 1, although wild type LuGST1-1

could act as a selenium-independent GPx for catalyzing

the decomposition of organic hydroperoxides such as

CuOOH and t-BuOOH, it did not catalyze the reduction

of hydrogen peroxides by using GSH. After incorporating

Sec into the active site of LuGST1-1, the generated

seleno-LuGST1-1 was endowed a significantly high GPx

activity comparable with natural GPx when hydrogen

peroxide was used as a substrate (Yu et al., 2005). In

addition, the GPx activities of seleno-LuGST1-1 for catalyz-

ing reduction of CuOOH and t-BuOOH were enhanced by

three orders of magnitude in comparison with those of wild

type LuGST1-1.

An initial slow rate of hydroperoxides consumption

was observed in the enzymatic reactions in which seleno-

LuGST1-1 was preincubated with hydroperoxides or

without any preincubation. This lag time was reduced

as GSH concentration was increased. When seleno-

LuGST1-1 was preincubated with excess GSH, no

obvious lag was observed. This observation implied that

the seleninic acid form of selenium lies off the main

catalytic cycle.

3.2. Kinetics of seleno-LuGST1-1-catalyzed reduction of

hydroperoxides by GSH

To probe the mechanism that seleno-LuGST1-1 applied

to catalyze the reduction of hydroperoxides by GSH,

detailed kinetic studies were carried out. Double-reciprocal

plots (Lineweaver–Burk plots) of initial velocity versus

substrate (GSH) concentration yielded families of linear

lines (Fig. 1).

The parallel lines corresponded to different concen-

trations of the catalyst and indicated that the velocities

increased with the concentration of seleno-LuGST1-1.

When the concentration of seleno-LuGST1-1 was fixed,

velocity rapidly increased with the increase of GSH

concentration in the initial phase and then approached to

a constant value as GSH concentration further increased.

A

0.4 0.8 1.2 1.6 2.00.00030

0.00035

0.00040

0.00045

0.00050

0.00055[C uOOH]= 0.5mM[C uOOH]= 1.0mM[C uOOH]= 2.0mM

B

0.4 0.8 1.2 1.6 2.0

0.00032

0.00036

0.00040

0.00044

0.00048

0.00052

1 / [CuOOH] (mM-1)

[GSH]=0.50mM[GSH]=1.00mM[GSH]=2.00mM

[E] 0

/ v0 (m

in)

[E] 0

/ v0 (m

in)

1 / [GSH] (mM-1)

Fig. 2. Double-reciprocal plots for the reduction of CuOOH by GSH

catalyzed by seleno-LuGST1-1 in 50 mM potassium phosphate buffer (pH

7.0) at 37 -C. Values are expressed as meansTSD (n�3). (A) [E]0/v0 vs. 1/

[GSH]; (B) [E]0/v0 vs. 1/[CuOOH].

0 1 2 3 4

0.1

0.2

0.3

1 / v

0 (µµ

M-1

min

)

1 / [GSH] (mM-1)

Fig. 1. Lineweaver–Burk plots obtained for the reduction of hydrogen

peroxide by GSH in the presence of different seleno-LuGST1-1 concen-

tration in 50 mM potassium phosphate buffer (pH 7.0) at 37 -C. The initial

hydrogen peroxide concentration was fixed to 0.5 mM. Values are

expressed as meansTSD (n�3). (g) [seleno-LuGST1-1]=0.0013 AM;

(>) [seleno-LuGST1-1]=0.0026 AM; (q) [seleno-LuGST1-1]=0.0052 AM;

(3) [seleno-LuGST1-1]=0.0104 AM.


At the same time, when the concentration of seleno-

LuGST1-1 was increased, the velocities became very high

for higher concentration of GSH. Similar to previous

studies on organic selenium derivatives (RSeSeR)-cata-

lyzed reduction of hydroperoxides by PhSH (Mugesh et

al., 2001b), these observations in our studies also suggest

that the intermediate ESeSG does certainly exist during

the catalytic cycle.

The kinetic experiments for seleno-LuGST1-1-cata-

lyzed-reduction of organic hydroperoxides were carried

out by varying one substrate’s concentration while keep-

ing the other constant. Saturation kinetics was observed

for the enzymatic peroxidase reaction at all the individual

concentration of GSH and t-BuOOH, H2O2 or CuOOH

investigated. Michaelis–Menten kinetics was observed for

all the substrates under all conditions investigated.

Double-reciprocal plots of initial velocity versus substrate

concentration at all the individual concentration revealed

the characteristic parallel lines of ping-pong mechanism

with at least one covalent intermediate (Figs. 2 and 3), in

analogy with our previous observation for H2O2 (Yu et

al., 2005). The data did not fit well to other mechanism,

such as sequential or equilibrium-ordered mechanisms.

In the light of ping-pong kinetic reaction, the following

equation for the initial velocity accounts for these plots

(Engel, 1981; Dalziel, 1969)

E½ �0v0

¼ U0 þUGSH

GSH½ � þUROOH

ROOH½ � ð3Þ

where v0 is the initial rate of the enzymatic reaction, and

[E]0 stands for the total concentration of the enzyme. U0 is

defined as the reciprocal of the maximum turnover number,

UROOH is defined as the reciprocal rate constant for the

ROOH-induced oxidation of isomerase EseH to EseOH,

UGSH is defined as the reciprocal rate constant for the sum

of the two reductive steps of the regeneration of the reduced

enzyme by GSH. These kinetic parameters were calculated

by fitting the experimental kinetic data (shown in Figs. 2-4)

to this Dalziel’s equation and the values were shown in

Table 2.

According to Scheme 1, the kinetic behavior of the

natural GPx could be described by Eq. (5) (Flohe, 1989):

E½ �0v0

¼ UGSH

GSH½ � þUROOH

ROOH½ � : ð4Þ

Eq. (5) is identical to Eq. (4) if it is considered that U0 in

Eq. (4) is negligible in the natural GPx cycle. This suggests

that seleno-LuGST1-1 may exactly follow the natural GPx

cycle.

In order to obtain the Michaelis kinetic parameters, the

Eq. (3) can be rearranged in the following form that bears

A

0

500

1000

1500

2000

2500

k cat

(min

-1)

k cat

(min

-1)

[GSH] (mM)B

0.0 0.5 1.0 1.5 2.0

0.0 0.5 1.0 1.5 2.0

0

500

1000

1500

2000

2500

3000

[ROOH] (mM)

Fig. 4. Plot of the apparent kcat (min�1) vs. substrate concentration for the

reduction of hydrogen peroxide with GSH catalyzed by seleno-LuGST1-1 in

50 mM potassium phosphate buffer (pH 7.0) at 37 -C. Values are expressed

as meansTSD (n�3). (A) kcat vs. [GSH] (mM), (g) H2O2 as the second

substrate; (>) CuOOH as the second substrate; (q) t-BuOOH as the second

substrate; (B) kcat vs. [ROOH] (mM), (g) H2O2 as the second substrate; (>)CuOOH as the second substrate; (q) t-BuOOH as the second substrate.

Table 2

Dalziel’s parameters for the seleno-LuGST1-1-catalyzed reduction of

hydroperoxides by GSHa

Hydroperoxide U0

(10�4 min�1)

UGSH

(10�7 M min)

UROOH

(10�7 M min)

H2O2 3.8T0.2 1.02T0.05 1.06T0.10

CuOOH 4.7T0.1 1.00T0.10 1.92T0.30

t-BuOOH 6.6T0.1 0.91T0.08 5.88T0.30a Reactions were carried out in 50 mM potassium phosphate buffer (pH

7.0) at 37 -C. The data in the table were calculated from the plots in Figs.

2–4 and are givenTS.D.

A

0.4 0.8 1.2 1.6 2.00.0006

0.0008

0.0010

0.0012

0.0014

0.0016

0.0018

0.0020

[E] 0

/ v 0

(m

in)

[E] 0

/ v 0

(m

in)

1 / [GSH] (mM-1)

[t-BuOOH]=0.2mM[t-BuOOH]=0.5mM[t-BuOOH]=1.0mM

B

1 32 4 50.0006

0.0008

0.0010

0.0012

0.0014

0.0016

0.0018

0.0020

1 / [t-BuOOH] (mM-1)

[GSH]=0.5mM [GSH]=1.0mM [GSH]=2.0mM

Fig. 3. Double-reciprocal plots for the reduction of t-BuOOH by GSH

catalyzed by seleno-LuGST1-1 in 50 mM potassium phosphate buffer (pH

7.0) at 37 -C. Values are expressed as meansTSD (n�3). (A) [E]0/v0 vs. 1/

[GSH]; (B) [E]0/v0 vs. 1/[t-BuOOH].


a more obvious relationship to the Michaelis–Menten

equation:

v0

E½ �0¼ kmax GSH½ � ROOH½ �

KROOH GSH½ � þ KGSH ROOH½ � þ GSH½ � ROOH½ �ð5Þ

where v0 is the initial rate of the enzymatic reaction, and

[E]0 stands for the total concentration of the enzyme, kmax

is a pseudo-first-order rate constant, the maximum rate of

reaction, KGSH and KROOH are the Michaelis–Menten

constant for GSH and ROOH, respectively. kmax/KGSH and

kmax/KGSH are the second-order rate constants. These

parameters were calculated from the Dalziel’s parameters

according to the following relationships: kmax=1/U0,

KGSH=UGSH/U0 and KROOH=UROOH/U0. The values of

these parameters were shown in Table 3.

It is important for seleno-LuGST1-1 that limited values

of kcat and Km were obtained for both GSH and H2O2 (Fig.

4). Similar results were also obtained when seleno-

LuGST1-1 catalyzed the reduction of CuOOH and t-

BuOOH by GSH.

4. Discussion

The similarity of the Dalziel equations for seleno-

LuGST1-1 and natural GPx suggests that they may follow

Table 3

Kinetic parameters for the seleno-LuGST1-1-catalyzed reduction of hydroperoxides by GSHa

Hydroperoxide kmax (min�1) KmROOH (10�4 M) kmax/KmROOH (M�1 min�1) KmGSH (10�4 M) kmax/KmGSH (M�1 min�1)

H2O2 2642T20 2.6T0.2 (9.4T0.1)106 2.6 T0.1 (9.8T0.2)106

CuOOH 2147T10 4.2T0.1 (5.2T0.3)106 2.2T0.3 (1.0T0.1)107

t-BuOOH 1518T30 9.1T0.3 (1.7T0.3)106 1.5T0.2 (1.1T0.2)107

a Reactions were carried out in 50 mM potassium phosphate buffer (pH 7.0) at 37 -C. Values are meansTS.D.


exactly the same catalytic cycle. This assumption was

further supported by the following observations. Firstly, as

has been mentioned above, the existence of EseSG in the

catalytic cycle of seleno-LuGST1-1 is identical to that of

Scheme 1 where the rate depends on the concentration of

ESeSG. Secondly, the observation of the lag time in the

reaction catalyzed by seleno-LuGST1-1 preincubated with

ROOH was in good analogy with those of the reactions

catalyzed by natural GPx in which (Scheme 1) the GPx

activity could be initiated with either the selenenyl sulfide

(ESeSG) or the seleninic acid (ESeO2H). The latter form

which lies off the main catalytic cycle may become the

majority at high concentrations of hydroperoxides. We

interpret the rate lag as the initial conversion of (ESeO2H)

to the selenenyl sulfide (ESeSG) by GSH. Thirdly, the

observation in our previous studies that seleno-LuGST1-1

lost GPx activity completely when treated with excess

iodoacetate acid in the presence of GSH suggested the

presentation of the enzyme-bound selenol (ESeH) in the

catalytic cycle (Yu et al., 2005). In addition, it could be

noted that all values for kmax/KGSH were identical, allowing

for experimental error, for the enzymatic reactions with

H2O2, CuOOH and t-BuOOH as shown in Table 3. This

suggests that the reaction between thiol and enzymic

intermediates is independent of the nature of hydroperoxides

as expected from the mechanism illustrated in Scheme 1.

According to the catalytic mechanism of naturally

occurring GPx, both of kcat and Km for hydroperoxides

at infinite concentration are linear functions of the

concentration of GSH and therefore Km and kmax are

indefinite. This fact implies that natural GPx could not be

saturated by GSH and thus U0 is negligible in Dalziel Eq.

(4) for U0 is the reciprocal of kmax (Ursini et al., 1995). In

contrast, the limited values of Km and kmax were obtained

for both GSH and hydroperoxides in the case of seleno-

LuGST1-1, indicating that this enzyme could be saturated

by both substrates and that U0 could not be omitted. These

differences suggest that there is no accumulation of a

Michaelis–Menten-type complex of oxidized enzyme and

reduced GSH (ESeSG) for naturally occurring GPx but

that such complex do build up in the case of seleno-

LuGST1-1. This might be explained by the different

affinities of these two enzymes to the second GSH

molecule. Naturally occurring GPx has a specific binding

site which could accommodate two GSH molecules. Apart

from some hydrogen bonds, two arginines (Arg) and one

lysine (Lys) could bind the two carboxylate groups of the

second GSH molecule and orient it into an adequate

position (Mugesh and Du Mont, 2001). Seleno-LuGST1-1,

however, has a binding site that could only specifically

bind one GSH molecule, which might make the rate of the

conjugation of seleno-LuGST1-1 to the second GSH

molecule much slower than that of the naturally occurring

GPx and therefore result in the accumulation of the

selenenyl sulfide adduct (ESeSG) in Scheme 1. This

suggests that it is possible to further enhance the catalytic

activity of seleno-LuGST1-1 by incorporating positive

charges such as Arg and Lys in the appropriate positions

of its active site to facilitate both binding and orienting of

the second GSH molecule through the rational redesign of

the enzyme.

Wild type LuGST1-1 exhibits selenium-independent

GPx activity against organic hydroperoxides just as many

other members of the GST family (Mannervik, 1985;

Jakoby, 1985; Tan and Board, 1996). A mechanism of

sequential addition of substrates followed by release of

products has been suggested for GST as opposed to the

ping-pong mechanism of GPx (Pierce and Tappel, 1978).

When GST acts upon organic hydroperoxides, GS-attacks an

electrophilic oxygen to form the unstable sulfenic acid of

glutathione (Eq. (6)) which then non-enzymatically reacts

with second equivalent of GSH to generate GSSG (Eq. (7))

(Prohaska, 1980).

ROOHþ GSH WGST

GSOH½ � þ ROH ð6Þ

GSOH½ � þ GSHWGSSGþ H2O: ð7Þ

It is important for the selenium-independent GPx activity

and the GST activity of LuGST1-1 that the active hydroxyl

group of Ser9 of LuGST1-1 could form hydrogen-bond with

the thiol group of GSH to stabilize the ionized GSH and

enhance the nucleophilicity of GSH (Caccuri et al., 1997).

Since the conversion of Ser9 of LuGST1-1 to Sec has

resulted in the significant decrease of GST activity as a

result of the removal of the active hydroxyl group (Yu et al.,

2005), the GPx activity of seleno-LuGST1-1 should have

also decreased to a large extend. However, the mutation of

LuGST1-1 to seleno-LuGST1-1 not only enhances the

enzymatic efficiency for decomposing organic hydroper-

oxides, but also endows the enzyme remarkably high GPx

activity to catalyze the reduction of hydrogen peroxide by

GSH. This could be attributed to the alteration of the

enzyme’s catalytic mechanism due to the single atom

substitution (from oxygen to selenium) in the catalytically

active residues. Selenium is characterized by the easy

change of its oxidation state and could be used to qualify


proteins containing Sec in the active sites for redox catalysis

(Mugesh and Du Mont, 2001; Mahmoud and Edens, 2003).

Most bacterial and mammalian selenoproteins could be

characterized as oxidoreductases and ping-pong mecha-

nism in analogy with that of GPx would be satisfied to

describe the mechanisms of most of these selenoproteins.

The first semi-synthetic selenoenzyme, selenosubtilisin,

also alters its chemical behavior and is afforded a novel

peroxidase activity with a ping-pong kinetics mechanism

(Bell et al., 1993; Bell and Hilvert, 1993). In the case of

seleno-LuGST1-1, selenium can be more easily oxidized

and reduced between valence state (II) and (IV) in

comparison to sulfur or oxygen (Wu et al., 1996).

Therefore, the lower redox potential of selenocysteine

compared with cysteine or serine is catalytically favorable

for the ESeHYESeOH conversion after which the

generated ESeOH is inclined to be reduced by reductant

such as GSH. And thus Sec enables seleno-LuGST1-1 the

favor for performing the GPx cycle for catalyzing

reduction of hydroperoxides by GSH rather than the

mechanism for the wild type GST. H2O2 is not a proper

substrate for GST and therefore its reduction by GSH

cannot be accelerated by GST through the reaction as

shown in Eq. (6). But it does act as a good oxidant for

selenol group in the catalytic cycle of GPx as shown in

Scheme 1. Thus, it is not surprising that seleno-LuGST1-1

is endowed with a high GPx activity toward H2O2 after

incorporation of Sec. At the same time, it is important to

note that when the catalytic cycle was altered from the

sequential one of GST (as shown in Eqs. (6) and (7)) to

the ping-pong mechanism of GPx (Scheme 1), the

enzymatic efficiency for decomposing organic hydroper-

oxides was greatly enhanced. This result suggests that the

selenium-dependent catalytic cycle of GPx is much more

efficient for the reduction of hydroperoxides by GSH than

the selenium-independent one performed by GST.

In conclusion, detailed kinetic studies of the GPx

activity of seleno-LuGST1-1 to various hydroperoxides

including H2O2, t-BuOOH, and CuOOH has been carried

out. In all the enzymatic reactions for the decomposition of

hydroperoxides investigated, the selenoenzyme exhibited a

ping-pong kinetic mechanism in analogy with that of the

natural GPx catalysis. In contrast to natural GPx, however,

saturation kinetics was observed for both GSH and hydro-

peroxides in the case of seleno-LuGST1-1. The selenium-

dependent ping-pong mechanism as opposed to the

sequential one of the wild type GST was assumed to result

from the intrinsic chemical properties of the incorporated

selenocysteine. The mutation of LuGST1-1 not only

enhanced the enzymatic efficiency for decomposing

organic hydroperoxides but also endowed the enzyme with

remarkably high GPx activity to catalyze the reduction of

hydrogen peroxide by GSH. This work extended our

understanding of biocatalysts and the evolution of GPx

itself and provided a basis for the development of more

efficient GPx mimics.

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