Vol. 182, No. 3, 1992 February 14, 1992
TVEOSINE-7
BIOCHEMICAL
IS
AN ESSENTIAL
HUMAN
CLASS
PI
UODIPICATION
Kwang-Hoon
RESIDUE
of
The Received
December
CATALYTIC
ACTIVITY
S-TEANSFEEASE:CHEMICAL
AND SITE-DIRECTED
MUTAGENESIS
Kong,
Motohiko
Biophysics
University
FOE TSE
GLUTATHIONE
and
Department
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1122-1129
of
Nishida,
Kenji
Biochemistry,
Tokyo,
Bunkyo-ku,
STUDIES
Hideshi
Inoue,
01
Takahashi
and
OF
Faculty Tokyo
of 113,
Science,
Japan
1991
26,
The glutathione (GSH)-conjugating activity of human S-Y: class Pi glutathione S-transferase (GSTx) toward l-chloro-2,4dinitrobenzene (CDNB) was significantly lowered by reaction with N-acetylimidazole, an 0-acetylating reagent for tyrosine residues. Further, the replacement of Tyr7 in GSTn, which is conserved in all cytosolic GSTs, with phenylalanine by sitedirected mutagenesis also lowered the activities toward CDNB and ethacrynic acid. The values of the mutant for both GSH and CDNB were almost equiva % ent to those of the wild type, while the V of the former was about 55-fold smaller than that of the l!%er. Therefore, Tyr7 is considered to be an essential residue for the catalytic activity of GSTn. 0 1992 Academic Press, Inc.
Glutathione of
multifunctional
conjugates of
Alpha,
between
Mu
about
1
reduced compounds
can
be
and
Pi,
properties
(4). 210-220
residues
0006-291X/92 Copyright All rights
grouped
amino are
CDNB,
are acid
conserved
should
EC 2.5.1.18)
catalyzing glutathione In
into
at to
homo-
their
residues. in
mammals
least
or
distinct and 5%
GST classes,
of GSTs
variety
cytosolic
structures
dytosolic
of
a wide
the
about
a family
formation
and
hetero-dimers
catalytic
subunits of
(2).
the
of amino
Although
be addressed.
used are: GST, glutathione 1-chloro-2,4-dinitrobenzene;
$1.50
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
three
Only all
are
the (GSH)
(l-3).
according
They
To whom correspondence
The abbreviations glutathione; acid.
(GSTS,
proteins,
electrophilic
isoenzymes
acid
S-transferases
1122
S-transferase; GSH, ETA, ethacrynic
Vol.
182,
No.
there of
3, 1992
are
many
reaction
is
and
essential
for
residues
in been
proved using
reported of
be
The
three-dimensional (17),
to
the
hydroxyl
bound
residue
GST
Pi that
the
of
GST the
to (GSTn)
be the
and
are cysteine in
studies reaction
GSH-
(5-ll),
mechanism
(12,13).
catalytic
is
also
in
all
of
this
important role by
from
They
by
were
activities
in
also other
expected
is
the
of
thiol in
Since
cytosolic
enzymatic
tyrosine
lie
Tyr7.
residue
modification
by
located
the to
of
glutathione-
reported
Accordingly,
classes
chemical
the
lung, group
group in
of
pig
sulfonate
Tyr7.
hydroxyl
conserved
studied
GST the
group of
is
presumed we
shows to
neighborhood
catalytic
participate
the
structure
class
al.
GSH
to
functions
the
residues
modification in
and
histidine to
mutagenesis
COMMUNICATIONS
(14-16).
sulfonate-bound et
? The
unessential
unessential
GSTs
of Which
suggested
chemical
site-directed
to
classes
by be
structures
mechanisms
activity GSTs,
to
the
the
RESEARCH
recognition.
catalytic class
reaction
studies
about
substrate
the
BIOPHYSICAL
on
known
the Pi
conjugating
AND
investigations
little
GSTs,
have
BIOCHEMICAL
group
of
immediate
this
tyrosine
function. and
adjacent
the GSTs
in
Reinemer
(2),
it
is
Therefore, human
class
Pi
site-directed
mutagenesis.
MATERIALS
AND METHODS
Materials. Wild-type human GSTlr was obtained by expression of a cloned cDNA, gifted by Prof. Muramatsu (18), in E. coli as described in the previous paper (12). GSH and l-chloro-2,4dinitrobenzene (CDNB) were purchased from Rohjin Co. and Wako Pure Chem. Ind., respectively. Ethacrynic acid and Shexylglutathione-Sepharose were obtained from Sigma. NAcetylimidazole and hydroxylamine were from Nakalai Tesque. Preparation of mutant enzymes. The oligonucleotide for mutagenesis of Tyr-7 into phenylalanine was designed as follows: 5'-ACCGTGGTCTTTTTCCCAGTT-3'. The synthesis the of oligonucleotide, construction of the mutant plasmid, and purification of the expressed transformation, expression, enzyme were performed as described in the previous paper (12). Enzyme assays. Protein concentration of the mutant Y7F was determined by using Protein assay reagent (Bio-Rad Lab.) and the wild-type enzyme as a standard protein. Assay of the enzyme activity was done as described in the previous paper (12). h& values were tentatively estimated assuming a steadyand 'max state mechanism. Reaction of N-acetslimidazole with GSTlr, Reaction of Nacetylimidazole with GSTJC was performed essentially as described by Masai et al. (19). The wild-type GSTn (0.51 PM) was incubated with 6.0 mM N-acetylimidazole in 20 mM potassium buffer (pH 7.0) at 20-C. N-Acetylimidaeole was phosphate dissolved in the same buffer just before use, and added to the Aliquots of the reaction mixture were used reaction mixtures.
1123
Vol.
182,
No.
BIOCHEMICAL
3, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
for enzyme assay. Treatment with hydroxylamine was done using the wild-type enzyme acetylated under the above conditions for 10 min. Hydroxylamine solution (2 M, pH 7.0) was prepared just before use. To 1 ml of the reaction mixture with Nacetylimidazole, 0.4 ml of the hydroxylamine solution was added and the mixture was incubated at 2O'C for 24 h, followed by dialysis against two changes of potassium phosphate buffer (20 for 8 h each at 4°C. mM, pH 7.0)
RESULTS Chemical
modification
GSTlr
was
reagent
for
also of
the
react
with
change
amino
in
The
remaining
when
6.0
Treatment
group and
was
of
is of
24
h regenerated
67%
of
inactivation
of
the
of
GSH,
Site-directed Y7F,
was
groups
but
not
mutagenesis prepared
by
the
GSTn the
0
1.
presence
Tyr7
in
10
10
PM
M)
not
in
the
I). of
GSTn,
The
mutant
15
(min)
Figure 1. The inactivation of wild type and mutant GSTxs by Nacetyliridazole. Wild-type GSTZ (0.51 PM), Y7F (5.7 PM) and C47S (0.78 PM) were incubated in the absence or in the presence of N-acetylimidazole (11,800-fold molar excess) in 20 mM potassium phosphate buffer (pH7.0) at 2O'C. Oand l , wild type without and with 6.0 mM N-acetylimidazole; hand A, C47S without and with 9.2 mM N-acetylimidazole;Oand n , Y7F without and with 67 mM N-acetylimidazole, respectively.
1124
for
shown).
(Table mutant
min
GSTlr.
blocked
mutagenesis.
6
13% at
(data
The
of
(1.4
CDNB
N-
incubation.
0.51
was of
GSTlr,
site-directed
Time
was
activity enzyme
course
activity
the
to
may
with
hydroxylamine
enzymic the
time
The
by
enzyme added
with
wild-type in
The
lowered wild-type
which
incubation
Fig.
was
of using
0’
by in
acetylating
residues, (19-22).
GSTn
Human
an
tyrosine
shown
acetylated
The
presence
thiol
N-acetylimidazole the
N-acetylimidazole.
significantly
activity mM
of
of
20°C
CDNB
with
N-acetylimidazole,
activity at
toward
GSTn
with
hydroxyl
acetylimidazole GSTK
of
incubated
Vol. 182, No. 3, 1992 Table
I.
BIOCHEMICAL
Protective inactivation
effects of GSTIS
Addition
Remaining activity
None GSH GSH
0.1 0.5
AND BIOPHYSICAL of substrate by N-acetyliridasole Addition
expressed
in
chromatography enzyme
was
coli
E.
on not
1.0 1.0 10.0
mM mM mM
was
isolated
and
by
the
of
mutant
(X)
9 8 GSTz
(0.51 After
purified
S-hexyl-GSH.
lowered
the
103
CDNB, wild type mM N-acetylimidazole. were assayed.
immobilized
on
Remaining activity
GSH CDNB CDNB
In the presence of GSH or PM) was incubated with 6.0 10 min, the remaining activities
GSTlr
binding
(%)
13 67 84
mM mM
RESEARCH COMMUNICATIONS
The
by
affinity
affinity
replacement
of
of
Tyr7
the with
phenylalanine. Kinetic
studies
activities
of
specific 8%
of $,
of
of wild
the
equivalent
both
type.
The
GSH
whose
highly
serine,
mutant
of
the
Table
II.
was
Specific for
less
were
type
(Table
mutants
of
activities GSA-conjugation
of
wild with
Specific activity
Values
type
76.6 1.0 are
means
f 1.5 + 0.1 f
of
an
of
Y7F
S.D.,
1 generally
A
(8-11)
type
type CDNB
1125
1.54 0.13 based
of
inhibitor,
mutant was
incubation
almost
replaced
1).
and mutant and ETA
+ 0.12 + 0.02 on
n>5.
C47S
with
(Fig.
Specific activity (pmol/min/mg)
100
to
was
However,
GSTxs
ETA Relative activity
III. that
inactivated.
(flmol/min/mg) Wild Y7F
2% of
GSTn,
residue wild
kinetic
Table
equivalent
only
CDNB
Enzyme
in
III).
through the
1% and
The
(IsO)
The
about
almost
was
Vmax
cysteine to
II).
were
shown
activity
inactivated similarly
Y7F
CDNB parameter
wild
ETA
are
conjugating
the
GSH-conjugating (Table
respectively.
the
reactive
was
by acetylimidazole
and
while
modification
CDNB and
substrates
inhibition of
The
assayed
type,
the
GSH-CDNB
that
were
toward
wild
type,
for to
Chemical
the
for wild
S-hexyl-GSH,
(12)s
toward
enzyme.
Y7F
Y7F
the
Y7F
values
those the
of
those of
the mutant
activities
parameters The
the
Relative activity
100 8
N-
Vol.
182, No. 3, 1992 Table
III.
BIOCHEMICAL
Enzymatic kinetic and inhibitory
AND BIOPHYSICAL parameters effect of
for GSH-CDNB S-hexyl-GSH
f&(mM) GSH 0.15 0.13
Values
are
CDNB
+ 0.01 + 0.02
means
0.82 1.02
+ S.D.,
conjugation
V max
Enzyme
Wild-type Y7F
RESEARCH COMMUNICATIONS
f +
150
(pmol/min) 0.04 0.07
generally
(PM)
164.1 3.0
based
+ 8.2 -+ 0.1
on
20.2 23.0
+ 0.5 f 0.4
n>3.
DISCUSSION GSTlc an
was
inactivated
0-acetylating
reagent
reagent
might
of
acetylated
the
also
acitivity.
This
attributed
to
and
its
the
hand,
seem
type
be
1).
out.
the
The
site-directed
mutagenesis
Therefore,
it
inactivation
tyrosine
or of
was
GSTn.
GSTs
in
crystallography Tyr7
in site
to it
(171,
the
is
of
the
enzyme-GSH
of
the
Cys47
does
by
as
active to
acetylation
of
cannot
be
N-
as
the
the
wild
the
other
completely
cysteine
residues by
that
to
the
in
studies
is
the
group
1126
in
importance
of
site-directed in
are all
present cytosolic
group
enzyme
is
to
hydroxyl
the group of
inactivation
of
resulted
residues
sulfonyl the thiol
The
the
conserved
the to
hydroxyl
complex.
acetylation
activity,
adjacent that
the
confirm
tyrosine
Tyr7
located the
41%
the
enzymatic
binds
presumed
neighborhood
of
N-acetylimidazole
order
since be
On
reactive,
similarly
possible by
In
that
groups.
(12,13).
only
glutathionesulfonate
S-
inactivate
demonstrated
Twelve
Moreover,
(2).
the
was
the
them,
because
inactivation
C47S,
of
most
performed.
Among
the
not
and highly to
inactivation
residues
GSTvt.
residues
mutagenesis in
seemed
residue
be
acetylation
non-essentiality activity
was
amino
reported
inactivated
the
the
groups,
to
the
that
caused
Treatment
inactivation
known
mutant
possibility
However,
a tyrosine
is
The
regenerated
N-acetylated
for
was
groups.
amino
were
GSH-conjugating
using
the
not
the
1).
0-acetyltyrosine
responsible
residues
ruled
of
GSTn
(12),
(Fig.
thiol
the
However,
since
(Fig.
cysteine
in
(8-11).
enzyme
and
deacetylate
modifications
acetylimidazole, wild-type
residues
that
but
N-acetylimidazole,
hydroxylamine
acetylation
Cys47
to
with
amino with
residues
completely
not
tyrosine
suggests
can
chemical
enzyme
for with
fact
acetylcysteine other
incubation
enzyme
hydroxylarine the
react
by
of Tyrl
of
shown GSH
the
by
X-ray
group lies
of also
in
the
active
of
the
enzyme
Vol.
182, No. 3, 1992
BIOCHEMICAL
by N-acetylimidazole GSH at
0.1
of the
acetylating
all that
in
-
was effectively
1 mM, which of
Therefore,
Tyr7
directed residues.
to
ETA (Table Tyr7
is
for
both
II).
necessary
wild-type
for
the
while
presence blocked
result
first
target roles
suggested
of
of
the
enzyme activity. the
the
the
site-
tyrosine
hydroxyl
The &,
equivalent
of
Vmax
the
at
responsible for the site of the enzyme.
that
almost
of
concentration
was not
This
the
suggests
GSH and CDNB were enzyme,
the
the
it
I).
the
with phenylalanine resulted toward CDNB and 92% loss
result
to
than
was GM-binding
investigate
of Tyr7 activity
This
(Table that
the
was selected
mutagenesis
The replacement of the specific
loss
CDNB residue in
in
mM), whereas
(6
was located
RESEARCH COMMUNICATIONS
blocked
was much lower
reagent
the presence the acetylated
inactivation
AND BIOPHYSICAL
group
values
to
former
in 99% toward
those
was about
of
of
Y7F
of
the
55-fold
smaller than that of the latter Tyr7 with phenylalanine scarcely
(Table III). The replacement of affected the inhibitory effect
of
(Table
III).
considered
to
S-hexyl-GSH
on
the
of
Tyr7
enzyme
group
catalytic or CDNB.
reaction of the enzyme but not in The result of chemical modification
is
consistent
with
effectively unlike
this
inactivated the
residue
wild-type
by
Chen et al. GST to catalysis
locates
hydroxyl
pKa value
1).
Thus,
for
the
GSH-binding
residues
may also
was
not N-acetylimidazole
Tyr7
in the
group of
the
of
Tyr7
phenolic
GSH Y7F so
seems to be the inactivation
site,
by
although
contribute
is
nucleophilicity although the
unknown.
hydroxyl
as in in
Since
group
to be protonated than acceptor.
might act as a general acid isomerase from P. testosteroni GSTlr actually participates
to
the some
in
of
the pKa value
general
of tyrosine
case
residue
at neutral pH and act Thus, the residue Tyr7
case of A'-3-ketosteroid (24), although whether Tyr7 in steroid isomerization (25) has
been examined.
conjugating
mutant
the
(23) mentioned that one major contribution of is to lower the pKa of the bound nucleophile.
is about 10, it is likely as a proton donor rather
not
(Fig.
in
the binding of of the mutant
with
may play a role in enhancing the group of GSH by such a mechanism,
of the the
incubation
the
participate
The
responsible
which
acetylation of the other extents to the inactivation.
Tyr7 thiol
conclusin.
enzyme
primarily
acetylation,
is
Consequently,
hydroxyl
the
However, at least enhancing the reaction, 1127
in the case of the GSH-CDNB elimination of the leaving
BIOCHEMICAL
Vol. 182, No. 3, 1992
group
by a general
important d-complex
the
through
protonation does not seem to be but not the decomposition, of a
formation,
intermediate was reported to be rate-limiting Tyr7 is presumed to contribute to deprotonation
Therefore, the thiol
group
group of Tyr7 deprotonation. contributes GSH in the also
of
GSH. This
may be possible
has an unusually Further studies
to lowering active site
During (26)
acid
because
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
low pKa value are necessary
the pKa of of GSTx.
preparation
of
this
reported
that
the
Alpha phenylalanine tyrosine this
Al-l,
the
counterpart
participate
in the
GST
over
the
lowers the residue
the
tbiol
replacement of
reaction
the
hydroxyl
to facilitate this to clarify how Tyr7 group
manuscript,
catalytic activity is considered
enzymatic
if
(23). of
of
the
Stenberg
bound et
al.
of Tyr8 in human class Tyr7 in with GSTn, of the enzyme. be essential to
Thus, and
mechanism of GSTs commonly
classes.
Acknowledstrent: We thank Professor Medicine, The University of Tokyo) placental GSTx cDNA.
Masami Muramatsu (Faculty of for providing us with human
REFERENCES 1. Mannervik, B. (1985) Adv. Ensymol. Rel. Areas mol. Biol., 57, 357-417. 2. Mannervik, B. & Danielson, U.H. (1988) CRC Crit. Rev. Biochem., 23, 283-337. 3. Pickett, C.B. t Lu, A.Y.H. (1989) Annu. Rev. Biocher., 58, 734-764. 4. Mannervik, B., Alin, P., Guthenberg, C., Jensson, H., Tahir, M.K., Warholm, M. & Jarnvall, H. (1985) Proc. Natl. Acad. Sci.
USA,
5. Awasthi, Biophys.
82,
Y.C., Res.
7202-7206.
Bhatnagar, Commun.,
A., 143,
& Singh,
S.V.
(1987)
Biochem.
965-970.
6. Van Ommen, B., Ploemen, J.H.T.M., Ruven, H.J., Vos, R.M.E., Bogaards, J.J.P., Van Berkel, W.J.H., & Van Bladeren, P.J. (1989)Eur. J. Biochem., 181, 423-429. 7. Andersson, C. & Horgenstern, R. (1990) Biochem. J, , 272, 479-484. 8. Tamai, K., Satoh, K., Tsuchida, S., Hatayama, I., Maki, T. & Sato, K. (1990) Biochem. Biophys. Res. Commun., 167, 331 -338. 9. Lo Bello, H., Petruzzelli, R., De Stefano, E., Tenedini, C., Barra, D. EL Federici, G. (1990) FEBS Lett., 263, 389-391. 10. Nishihara, T., Maeda, H., Okamoto, K., Oshida, T., Mizoguchi, T. & Terada, T. (1991) Biochem. Biophys. Res. Commun., 174, 580-585. 11. Desideri, A., Caccuri, A.M., Poligio, F., Bastoni, R. & Federici, G. (1991) J. Biochem. Chem., 266, 2063-2066. 12. Kong, K.-H., Inoue, H. & Takahashi. K. (1991) Biochem. Biophys. Res. Commun., 181, 748-755. 1128
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Tamai, K., Shen, H., Tsuchida, S., Hatayama, I., Satoh, K., Yasui, A., Oikawa, A., & Sato, K. (1991) Biochem. Biophys. Res. Commun. , 179, 790-797. Wang, R.W., Newton, D.J., Pickett, C.B. & Lu, A.Y.H. (1991) Arch. Biochem. Biophys., 286, 574-578. Zhang, P., Graminski, G.F. & Armstrong, R.N. (1991) J. Biol. Chem., 266, 19475-1947s. Widersten, M., Holmstrtim, E. & Mannervik, B. (1991) FEBS Lett., 293, 156-159. Reinemer, P., Dirr, H. W., Ladenstein, R., Schaffer, J., Gallay, 0. t Huber, R. (1991) EMBO J., 10, 1997-2005. Kano, T., Sakai, M. and Muramatsu, M. (1987) Cancer Res., 47, 5626-5630. Kasai, H., Takahashi, K. & Ando, T. (1977) J. Biochem. (Tokyo), 81, 1751-1758. Riordan, J.F. & Vallee, B.L., (1972) Methods Enzymol., 25, 500-506. Simpson, R.T., & Vallee, B.L. (1963) Riordan, J.F. Biochemistry, 2, 616-622. Lundblad, R.L., Harrison, J.H. & Mann, K.G. (1973) Biochemistry, 12, 409-413. Chen, W.-J., Graminski, G.F. & Armstrong, R.N. (1988) Biochemistry, 27, 647-654. Xue, L., Talalay, P. & Mildvan, A.S. (1991) Biochemistry, 30, 10858-10865. Benson, A.M., Talalay, P., Keen, J.H. & Jakoby, W.B. (1977) Proc. Natl. Acad. Sci. USA, 74, 158-162. Stenberg, G., Board, P.G. & Mannervik, b. (1991) FEBS Lett., 293, 153-155.
1129
TVEOSINE-7
BIOCHEMICAL
IS
AN ESSENTIAL
HUMAN
CLASS
PI
UODIPICATION
Kwang-Hoon
RESIDUE
of
The Received
December
CATALYTIC
ACTIVITY
S-TEANSFEEASE:CHEMICAL
AND SITE-DIRECTED
MUTAGENESIS
Kong,
Motohiko
Biophysics
University
FOE TSE
GLUTATHIONE
and
Department
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1122-1129
of
Nishida,
Kenji
Biochemistry,
Tokyo,
Bunkyo-ku,
STUDIES
Hideshi
Inoue,
01
Takahashi
and
OF
Faculty Tokyo
of 113,
Science,
Japan
1991
26,
The glutathione (GSH)-conjugating activity of human S-Y: class Pi glutathione S-transferase (GSTx) toward l-chloro-2,4dinitrobenzene (CDNB) was significantly lowered by reaction with N-acetylimidazole, an 0-acetylating reagent for tyrosine residues. Further, the replacement of Tyr7 in GSTn, which is conserved in all cytosolic GSTs, with phenylalanine by sitedirected mutagenesis also lowered the activities toward CDNB and ethacrynic acid. The values of the mutant for both GSH and CDNB were almost equiva % ent to those of the wild type, while the V of the former was about 55-fold smaller than that of the l!%er. Therefore, Tyr7 is considered to be an essential residue for the catalytic activity of GSTn. 0 1992 Academic Press, Inc.
Glutathione of
multifunctional
conjugates of
Alpha,
between
Mu
about
1
reduced compounds
can
be
and
Pi,
properties
(4). 210-220
residues
0006-291X/92 Copyright All rights
grouped
amino are
CDNB,
are acid
conserved
should
EC 2.5.1.18)
catalyzing glutathione In
into
at to
homo-
their
residues. in
mammals
least
or
distinct and 5%
GST classes,
of GSTs
variety
cytosolic
structures
dytosolic
of
a wide
the
about
a family
formation
and
hetero-dimers
catalytic
subunits of
(2).
the
of amino
Although
be addressed.
used are: GST, glutathione 1-chloro-2,4-dinitrobenzene;
$1.50
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
three
Only all
are
the (GSH)
(l-3).
according
They
To whom correspondence
The abbreviations glutathione; acid.
(GSTS,
proteins,
electrophilic
isoenzymes
acid
S-transferases
1122
S-transferase; GSH, ETA, ethacrynic
Vol.
182,
No.
there of
3, 1992
are
many
reaction
is
and
essential
for
residues
in been
proved using
reported of
be
The
three-dimensional (17),
to
the
hydroxyl
bound
residue
GST
Pi that
the
of
GST the
to (GSTn)
be the
and
are cysteine in
studies reaction
GSH-
(5-ll),
mechanism
(12,13).
catalytic
is
also
in
all
of
this
important role by
from
They
by
were
activities
in
also other
expected
is
the
of
thiol in
Since
cytosolic
enzymatic
tyrosine
lie
Tyr7.
residue
modification
by
located
the to
of
glutathione-
reported
Accordingly,
classes
chemical
the
lung, group
group in
of
pig
sulfonate
Tyr7.
hydroxyl
conserved
studied
GST the
group of
is
presumed we
shows to
neighborhood
catalytic
participate
the
structure
class
al.
GSH
to
functions
the
residues
modification in
and
histidine to
mutagenesis
COMMUNICATIONS
(14-16).
sulfonate-bound et
? The
unessential
unessential
GSTs
of Which
suggested
chemical
site-directed
to
classes
by be
structures
mechanisms
activity GSTs,
to
the
the
RESEARCH
recognition.
catalytic class
reaction
studies
about
substrate
the
BIOPHYSICAL
on
known
the Pi
conjugating
AND
investigations
little
GSTs,
have
BIOCHEMICAL
group
of
immediate
this
tyrosine
function. and
adjacent
the GSTs
in
Reinemer
(2),
it
is
Therefore, human
class
Pi
site-directed
mutagenesis.
MATERIALS
AND METHODS
Materials. Wild-type human GSTlr was obtained by expression of a cloned cDNA, gifted by Prof. Muramatsu (18), in E. coli as described in the previous paper (12). GSH and l-chloro-2,4dinitrobenzene (CDNB) were purchased from Rohjin Co. and Wako Pure Chem. Ind., respectively. Ethacrynic acid and Shexylglutathione-Sepharose were obtained from Sigma. NAcetylimidazole and hydroxylamine were from Nakalai Tesque. Preparation of mutant enzymes. The oligonucleotide for mutagenesis of Tyr-7 into phenylalanine was designed as follows: 5'-ACCGTGGTCTTTTTCCCAGTT-3'. The synthesis the of oligonucleotide, construction of the mutant plasmid, and purification of the expressed transformation, expression, enzyme were performed as described in the previous paper (12). Enzyme assays. Protein concentration of the mutant Y7F was determined by using Protein assay reagent (Bio-Rad Lab.) and the wild-type enzyme as a standard protein. Assay of the enzyme activity was done as described in the previous paper (12). h& values were tentatively estimated assuming a steadyand 'max state mechanism. Reaction of N-acetslimidazole with GSTlr, Reaction of Nacetylimidazole with GSTJC was performed essentially as described by Masai et al. (19). The wild-type GSTn (0.51 PM) was incubated with 6.0 mM N-acetylimidazole in 20 mM potassium buffer (pH 7.0) at 20-C. N-Acetylimidaeole was phosphate dissolved in the same buffer just before use, and added to the Aliquots of the reaction mixture were used reaction mixtures.
1123
Vol.
182,
No.
BIOCHEMICAL
3, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
for enzyme assay. Treatment with hydroxylamine was done using the wild-type enzyme acetylated under the above conditions for 10 min. Hydroxylamine solution (2 M, pH 7.0) was prepared just before use. To 1 ml of the reaction mixture with Nacetylimidazole, 0.4 ml of the hydroxylamine solution was added and the mixture was incubated at 2O'C for 24 h, followed by dialysis against two changes of potassium phosphate buffer (20 for 8 h each at 4°C. mM, pH 7.0)
RESULTS Chemical
modification
GSTlr
was
reagent
for
also of
the
react
with
change
amino
in
The
remaining
when
6.0
Treatment
group and
was
of
is of
24
h regenerated
67%
of
inactivation
of
the
of
GSH,
Site-directed Y7F,
was
groups
but
not
mutagenesis prepared
by
the
GSTn the
0
1.
presence
Tyr7
in
10
10
PM
M)
not
in
the
I). of
GSTn,
The
mutant
15
(min)
Figure 1. The inactivation of wild type and mutant GSTxs by Nacetyliridazole. Wild-type GSTZ (0.51 PM), Y7F (5.7 PM) and C47S (0.78 PM) were incubated in the absence or in the presence of N-acetylimidazole (11,800-fold molar excess) in 20 mM potassium phosphate buffer (pH7.0) at 2O'C. Oand l , wild type without and with 6.0 mM N-acetylimidazole; hand A, C47S without and with 9.2 mM N-acetylimidazole;Oand n , Y7F without and with 67 mM N-acetylimidazole, respectively.
1124
for
shown).
(Table mutant
min
GSTlr.
blocked
mutagenesis.
6
13% at
(data
The
of
(1.4
CDNB
N-
incubation.
0.51
was of
GSTlr,
site-directed
Time
was
activity enzyme
course
activity
the
to
may
with
hydroxylamine
enzymic the
time
The
by
enzyme added
with
wild-type in
The
lowered wild-type
which
incubation
Fig.
was
of using
0’
by in
acetylating
residues, (19-22).
GSTn
Human
an
tyrosine
shown
acetylated
The
presence
thiol
N-acetylimidazole the
N-acetylimidazole.
significantly
activity mM
of
of
20°C
CDNB
with
N-acetylimidazole,
activity at
toward
GSTn
with
hydroxyl
acetylimidazole GSTK
of
incubated
Vol. 182, No. 3, 1992 Table
I.
BIOCHEMICAL
Protective inactivation
effects of GSTIS
Addition
Remaining activity
None GSH GSH
0.1 0.5
AND BIOPHYSICAL of substrate by N-acetyliridasole Addition
expressed
in
chromatography enzyme
was
coli
E.
on not
1.0 1.0 10.0
mM mM mM
was
isolated
and
by
the
of
mutant
(X)
9 8 GSTz
(0.51 After
purified
S-hexyl-GSH.
lowered
the
103
CDNB, wild type mM N-acetylimidazole. were assayed.
immobilized
on
Remaining activity
GSH CDNB CDNB
In the presence of GSH or PM) was incubated with 6.0 10 min, the remaining activities
GSTlr
binding
(%)
13 67 84
mM mM
RESEARCH COMMUNICATIONS
The
by
affinity
affinity
replacement
of
of
Tyr7
the with
phenylalanine. Kinetic
studies
activities
of
specific 8%
of $,
of
of wild
the
equivalent
both
type.
The
GSH
whose
highly
serine,
mutant
of
the
Table
II.
was
Specific for
less
were
type
(Table
mutants
of
activities GSA-conjugation
of
wild with
Specific activity
Values
type
76.6 1.0 are
means
f 1.5 + 0.1 f
of
an
of
Y7F
S.D.,
1 generally
A
(8-11)
type
type CDNB
1125
1.54 0.13 based
of
inhibitor,
mutant was
incubation
almost
replaced
1).
and mutant and ETA
+ 0.12 + 0.02 on
n>5.
C47S
with
(Fig.
Specific activity (pmol/min/mg)
100
to
was
However,
GSTxs
ETA Relative activity
III. that
inactivated.
(flmol/min/mg) Wild Y7F
2% of
GSTn,
residue wild
kinetic
Table
equivalent
only
CDNB
Enzyme
in
III).
through the
1% and
The
(IsO)
The
about
almost
was
Vmax
cysteine to
II).
were
shown
activity
inactivated similarly
Y7F
CDNB parameter
wild
ETA
are
conjugating
the
GSH-conjugating (Table
respectively.
the
reactive
was
by acetylimidazole
and
while
modification
CDNB and
substrates
inhibition of
The
assayed
type,
the
GSH-CDNB
that
were
toward
wild
type,
for to
Chemical
the
for wild
S-hexyl-GSH,
(12)s
toward
enzyme.
Y7F
Y7F
the
Y7F
values
those the
of
those of
the mutant
activities
parameters The
the
Relative activity
100 8
N-
Vol.
182, No. 3, 1992 Table
III.
BIOCHEMICAL
Enzymatic kinetic and inhibitory
AND BIOPHYSICAL parameters effect of
for GSH-CDNB S-hexyl-GSH
f&(mM) GSH 0.15 0.13
Values
are
CDNB
+ 0.01 + 0.02
means
0.82 1.02
+ S.D.,
conjugation
V max
Enzyme
Wild-type Y7F
RESEARCH COMMUNICATIONS
f +
150
(pmol/min) 0.04 0.07
generally
(PM)
164.1 3.0
based
+ 8.2 -+ 0.1
on
20.2 23.0
+ 0.5 f 0.4
n>3.
DISCUSSION GSTlc an
was
inactivated
0-acetylating
reagent
reagent
might
of
acetylated
the
also
acitivity.
This
attributed
to
and
its
the
hand,
seem
type
be
1).
out.
the
The
site-directed
mutagenesis
Therefore,
it
inactivation
tyrosine
or of
was
GSTn.
GSTs
in
crystallography Tyr7
in site
to it
(171,
the
is
of
the
enzyme-GSH
of
the
Cys47
does
by
as
active to
acetylation
of
cannot
be
N-
as
the
the
wild
the
other
completely
cysteine
residues by
that
to
the
in
studies
is
the
group
1126
in
importance
of
site-directed in
are all
present cytosolic
group
enzyme
is
to
hydroxyl
the group of
inactivation
of
resulted
residues
sulfonyl the thiol
The
the
conserved
the to
hydroxyl
complex.
acetylation
activity,
adjacent that
the
confirm
tyrosine
Tyr7
located the
41%
the
enzymatic
binds
presumed
neighborhood
of
N-acetylimidazole
order
since be
On
reactive,
similarly
possible by
In
that
groups.
(12,13).
only
glutathionesulfonate
S-
inactivate
demonstrated
Twelve
Moreover,
(2).
the
was
the
them,
because
inactivation
C47S,
of
most
performed.
Among
the
not
and highly to
inactivation
residues
GSTvt.
residues
mutagenesis in
seemed
residue
be
acetylation
non-essentiality activity
was
amino
reported
inactivated
the
the
groups,
to
the
that
caused
Treatment
inactivation
known
mutant
possibility
However,
a tyrosine
is
The
regenerated
N-acetylated
for
was
groups.
amino
were
GSH-conjugating
using
the
not
the
1).
0-acetyltyrosine
responsible
residues
ruled
of
GSTn
(12),
(Fig.
thiol
the
However,
since
(Fig.
cysteine
in
(8-11).
enzyme
and
deacetylate
modifications
acetylimidazole, wild-type
residues
that
but
N-acetylimidazole,
hydroxylamine
acetylation
Cys47
to
with
amino with
residues
completely
not
tyrosine
suggests
can
chemical
enzyme
for with
fact
acetylcysteine other
incubation
enzyme
hydroxylarine the
react
by
of Tyrl
of
shown GSH
the
by
X-ray
group lies
of also
in
the
active
of
the
enzyme
Vol.
182, No. 3, 1992
BIOCHEMICAL
by N-acetylimidazole GSH at
0.1
of the
acetylating
all that
in
-
was effectively
1 mM, which of
Therefore,
Tyr7
directed residues.
to
ETA (Table Tyr7
is
for
both
II).
necessary
wild-type
for
the
while
presence blocked
result
first
target roles
suggested
of
of
the
enzyme activity. the
the
the
site-
tyrosine
hydroxyl
The &,
equivalent
of
Vmax
the
at
responsible for the site of the enzyme.
that
almost
of
concentration
was not
This
the
suggests
GSH and CDNB were enzyme,
the
the
it
I).
the
with phenylalanine resulted toward CDNB and 92% loss
result
to
than
was GM-binding
investigate
of Tyr7 activity
This
(Table that
the
was selected
mutagenesis
The replacement of the specific
loss
CDNB residue in
in
mM), whereas
(6
was located
RESEARCH COMMUNICATIONS
blocked
was much lower
reagent
the presence the acetylated
inactivation
AND BIOPHYSICAL
group
values
to
former
in 99% toward
those
was about
of
of
Y7F
of
the
55-fold
smaller than that of the latter Tyr7 with phenylalanine scarcely
(Table III). The replacement of affected the inhibitory effect
of
(Table
III).
considered
to
S-hexyl-GSH
on
the
of
Tyr7
enzyme
group
catalytic or CDNB.
reaction of the enzyme but not in The result of chemical modification
is
consistent
with
effectively unlike
this
inactivated the
residue
wild-type
by
Chen et al. GST to catalysis
locates
hydroxyl
pKa value
1).
Thus,
for
the
GSH-binding
residues
may also
was
not N-acetylimidazole
Tyr7
in the
group of
the
of
Tyr7
phenolic
GSH Y7F so
seems to be the inactivation
site,
by
although
contribute
is
nucleophilicity although the
unknown.
hydroxyl
as in in
Since
group
to be protonated than acceptor.
might act as a general acid isomerase from P. testosteroni GSTlr actually participates
to
the some
in
of
the pKa value
general
of tyrosine
case
residue
at neutral pH and act Thus, the residue Tyr7
case of A'-3-ketosteroid (24), although whether Tyr7 in steroid isomerization (25) has
been examined.
conjugating
mutant
the
(23) mentioned that one major contribution of is to lower the pKa of the bound nucleophile.
is about 10, it is likely as a proton donor rather
not
(Fig.
in
the binding of of the mutant
with
may play a role in enhancing the group of GSH by such a mechanism,
of the the
incubation
the
participate
The
responsible
which
acetylation of the other extents to the inactivation.
Tyr7 thiol
conclusin.
enzyme
primarily
acetylation,
is
Consequently,
hydroxyl
the
However, at least enhancing the reaction, 1127
in the case of the GSH-CDNB elimination of the leaving
BIOCHEMICAL
Vol. 182, No. 3, 1992
group
by a general
important d-complex
the
through
protonation does not seem to be but not the decomposition, of a
formation,
intermediate was reported to be rate-limiting Tyr7 is presumed to contribute to deprotonation
Therefore, the thiol
group
group of Tyr7 deprotonation. contributes GSH in the also
of
GSH. This
may be possible
has an unusually Further studies
to lowering active site
During (26)
acid
because
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
low pKa value are necessary
the pKa of of GSTx.
preparation
of
this
reported
that
the
Alpha phenylalanine tyrosine this
Al-l,
the
counterpart
participate
in the
GST
over
the
lowers the residue
the
tbiol
replacement of
reaction
the
hydroxyl
to facilitate this to clarify how Tyr7 group
manuscript,
catalytic activity is considered
enzymatic
if
(23). of
of
the
Stenberg
bound et
al.
of Tyr8 in human class Tyr7 in with GSTn, of the enzyme. be essential to
Thus, and
mechanism of GSTs commonly
classes.
Acknowledstrent: We thank Professor Medicine, The University of Tokyo) placental GSTx cDNA.
Masami Muramatsu (Faculty of for providing us with human
REFERENCES 1. Mannervik, B. (1985) Adv. Ensymol. Rel. Areas mol. Biol., 57, 357-417. 2. Mannervik, B. & Danielson, U.H. (1988) CRC Crit. Rev. Biochem., 23, 283-337. 3. Pickett, C.B. t Lu, A.Y.H. (1989) Annu. Rev. Biocher., 58, 734-764. 4. Mannervik, B., Alin, P., Guthenberg, C., Jensson, H., Tahir, M.K., Warholm, M. & Jarnvall, H. (1985) Proc. Natl. Acad. Sci.
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1129
