From the Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716
Received for publication, September 7, 2000, and in revised form, October 10, 2000
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ABSTRACT |
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Rat liver glutathione S-transferase,
isozyme 1-1, catalyzes the glutathione-dependent
isomerization of Glutathione S-transferases
(GST)1 (EC 2.5.1.18)
constitute a family of detoxification enzymes that are involved in the
metabolism of endogenous and xenobiotic compounds (1-4). They catalyze
the conjugation reaction of glutathione to a wide variety of
electrophilic substrates. These conjugation products are more
water-soluble than the xenobiotic substrates, and they can be further
degraded or transported out of the cell. Glutathione
S-transferases have been found in elevated levels within
cancerous tumors and have been implicated in the development of
resistance to anti-cancer drugs (5). The cytosolic enzymes are now
grouped into seven classes and within a particular class they can exist
as either homo- or heterodimers (1). There are crystal structures to represent most of the classes (6-12). Each subunit of the dimer contains a glutathione-binding site and a xenobiotic site that can
accommodate a wide variety of compounds.
Isozyme 1-1,2 a member of the
Materials--
Frozen Harlan Sprague-Dawley rat livers were
purchased from Pel Freez Biologicals, glutathione,
S-hexylglutathione, S-hexylglutathione-Sepharose, S-methylglutathione, Sephadex G-50, iodoacetic acid,
Enzyme Preparation--
Glutathione S-transferase
isozyme 1-1 was purified from rat livers using affinity column
chromatography on S-hexylglutathione-Sepharose (19). Values
of Synthesis of
17
The 17
For the radioactively labeled compound, the specific radioactivity was
2.17 × 1011 cpm/mol. The product has a UV absorption
spectrum with a maximum at 260 nm and a shoulder at 270 nm. The
extinction coefficient at 260 nm was measured to be 1810 M Enzymatic Assays--
Enzymatic activity was measured by using a
Hewlett Packard 8453 UV-VIS Spectrophotometer and monitoring the
formation of the glutathione (2.5 mM in assay) and
1-chloro-2,4-dinitrobenzene (1 mM in assay) conjugate at
340 nm ( Reaction of 17 Measurement of Incorporation of 17 Preparation and Separation of Proteolytic Digest of Modified
Glutathione S-Transferase--
Glutathione S-transferase
(0.2 mg/ml) was incubated with 500 µM
14C-labeled 17
The enzyme was solubilized by adding 250 µl of 8 M urea
in 10 mM ammonium bicarbonate, pH 8.0, and incubating at
37 °C for 1 h. The solution was then diluted with 10 mM ammonium bicarbonate to bring the final concentration of
urea to 2 M. Chymotrypsin was added (10% w/w) at 2 h
intervals while incubating at 37 °C. The ester bond between the
iodoacetic acid and estradiol-3-sulfate was subsequently hydrolyzed by
adding 2 N NaOH to yield 0.2 N NaOH and then
incubating the enzyme digest at 25 °C for 2 h. The solution was
then neutralized by adding HCl to yield 0.2 N. The solution
was filtered through a 0.45 µM filter, with no loss of radioactivity and was subjected to HPLC.
The chymotryptic peptides were fractionated by a Varian 5000 LC
equipped with a Vydac C18 reverse-phase column equilibrated with Solvent A (0.1% trifluoroacetic acid in water). At a flow rate of
1 ml/min, the peptides were separated by a linear gradient from 0% to
20% Solvent B (0.1% trifluoroacetic acid in acetonitrile) in 100 min
followed by a linear gradient to 100% Solvent B in 30 min. The eluate
was monitored by A220, and 1-ml fractions were collected. An aliquot (300 µl) from each fraction was added to 5 ml
of Liquiscint to test for radioactivity.
Sequence Determination of Separated Peptides--
The amino acid
sequences of purified peptides were determined on an Applied Biosystems
model 470A gas phase protein/peptide sequencer, equipped with a model
120A phenylthiohydantoin analyzer.
Molecular Modeling--
Molecular modeling was conducted using
the Insight II modeling package from Molecular Simulations, Inc. on an
Indigo 2 work station from Silicon Graphics. The model of rat GST 1-1 was constructed as described previously (19) based on the known crystal
structure of human liver isozyme 1-1 (1GUH). The structure of 17 Inactivation of Rat Liver Glutathione S-transferase 1-1 by
17 Concentration Dependence of the Rate of Inactivation--
GST 1-1 (0.2 mg/ml, 7.8 µM enzyme subunits) was incubated with
20-300 µM of 17 Effect of Ligands on the Inactivation Rate of GST 1-1 by
17 Incorporation of Radioactive 17 Isolation and Characterization of Chymotryptic Peptides from
17 17 Upon maximum inactivation, about 0.5 mol of reagent is incorporated per
mol enzyme subunit or 1 mol of 17 In the case of glutathionyl
S-[4-(succinimidyl)benzophenone), only one subunit is
modified, yet the enzyme is completely inactivated. The modification of
one subunit thus can abolish the enzyme activity of both subunits and,
because this label does not occupy the nonsubstrate site, the
inhibition is probably the result of a subtle conformational change
rather than a physical barrier to the binding of the substrate (28).
There is also complete inactivation by the aflatoxin conjugate, although in this case, the bound conjugate extends into the cleft and
therefore may be inhibiting completely either because it is blocking
access to the active site of the unmodified subunit or because it
induces a conformational change (25).
In the present case, maximum reaction with 17 A homology model for the rat 1-1 isozyme was generated from the crystal
structure of the human glutathione S-transferase 1-1. The
reagent was manually docked into the model based on an energy-minimized structure of 175-androstene-3,17-dione and also binds
steroid sulfates at a nonsubstrate inhibitory steroid site.
17
-Iodoacetoxy-estradiol-3-sulfate, a reactive steroid analogue,
produces a time-dependent inactivation of this glutathione
S-transferase to a limit of 60% residual activity. The
rate constant for inactivation (kobs) exhibits
a nonlinear dependence on reagent concentration with
KI = 71 µM and kmax = 0.0133 min
1. Complete
protection against inactivation is provided by
17
-estradiol-3,17-disulfate, whereas
5-androstene-3,17-dione and
S-methylglutathione have little effect on
kobs. These results indicate that
17
-iodoacetoxy-estradiol-3-sulfate reacts as an affinity label of
the nonsubstrate steroid site rather than of the substrate sites
occupied by
5-androstene-3,17-dione or glutathione. Loss
of activity occurs concomitant with incorporation of about 1 mol
14C-labeled reagent/mol enzyme dimer when the enzyme
is maximally inactivated. Isolation of the labeled peptide from the
chymotryptic digest shows that Cys17 is the only enzymic
amino acid modified. Covalent modification of Cys17 by
17
-iodoacetoxy-estradiol-3-sulfate on subunit A prevents reaction of
the steroid analogue with subunit B. These results and examination of
the crystal structure of the enzyme suggest that the interaction
between the two subunits of glutathione S-transferase 1-1, and the electrostatic attraction between the 3-sulfate of the reagent
and Arg14 of subunit B, are important in binding steroid
sulfates at the nonsubstrate steroid binding site and in determining
the specificity of this affinity label.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
class, efficiently catalyzes the isomerization reaction of
5-androstene-3,17-dione to
4-androstene-3,17-dione, which it binds at the substrate
steroid site (13). In addition to this site, isozyme 1-1 also has a nonsubstrate steroid binding site that is located in the cleft between
the two subunits (14, 15). This site has been proposed to fulfill a
transport function (5) or to act in controlling levels of steroids in
target organs (16). The nonsubstrate site has a preference for steroid
sulfates, which is illustrated by the more potent inhibitory effect of
17
-estradiol-3,17-disulfate as compared with that of
17
-estradiol. However, previous work in this laboratory (aimed at
locating the nonsubstrate site) used the affinity label
3
-(iodoacetoxy)dehydroisoandrosterone (3
-IDA) (shown in
Fig. 1), which is structurally related
both to substrates of the enzyme, such as
5-androstene-3,17-dione, and to inhibitors of the
enzyme, such as
5-androstene-3
,17
-diol disulfate
and 17
-estradiol-3,17-disulfate. The 3
-IDA modified
Cys17 and Cys111 equally with an incorporation
of 1 mol of reagent/mol enzyme subunit; analysis of molecular models
suggested that the binding site of 3
-IDA is located in the cleft
between the subunits (15). Based on the previous data, we have now
designed a more specific affinity label for the nonsubstrate steroid
site: 17
-iodoacetoxy-estradiol-3-sulfate (17
-IES). This new
compound features the negatively charged sulfate that should enhance
and direct its binding and a reactive iodoacetoxy group at a position
at the opposite end of the molecule from that of 3
-IDA (Fig. 1). The
iodide can be displaced from the iodoacetoxy group by nucleophilic
attack by the side chains of several amino acids including Cys, Asp,
Lys, Met, and His (17). In this paper, we demonstrate that this
affinity label reacts specifically with Cys17 at a single
subunit of the enzyme dimer. Molecular modeling studies support the
location of the nonsubstrate binding site within the cleft and the
contribution of the sulfate moiety in orienting the ligand within the
cleft. A preliminary version of this work has been presented (18).
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Fig. 1.
Steroids that bind to glutathione
S-transferase, isozyme 1-1. 5-Androstene-3,17-dione (substrate),
17
-estradiol-3,17-disulfate (reversible inhibitor),
3
-(iodoacetoxy)dehydroisoandrosterone (affinity label), and
17
-iodoacetoxy-estradiol-3-sulfate (affinity label) are
shown.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chymotrypsin, 17
-estradiol-3,17-disulfate,
17
-estradiol-3-sulfate,
N,N'-dicyclohexylcarbodiimide, and
1-chloro-2,4-dinitrobenzene were purchased from Sigma.
17
-Estradiol-17-sulfate and
5-androstene-3,17-dione
were provided by Steraloids, Inc., and [1-14C]iodoacetic
acid was purchased from Moravek Biochemicals. Bio-Rad Laboratories
provided Protein Assay Dye Reagent, and Liquiscint was purchased from
National Diagnostics.
270 nm = 22,000 M
1
cm
1 (20) and molecular weight of 25,500 per subunit (2)
for GST 1-1 were used to calculate the enzyme concentration.
-Iodoacetoxy-estradiol-3-sulfate--
17
-IES was synthesized
from 17
-estradiol-3-sulfate and iodoacetic acid by procedures based
on the method of Pons et al. (21). One molar equivalent of
17
-estradiol-3-sulfate, 1.1 molar equivalents of iodoacetic acid,
and 2 molar equivalents of dicyclohexylcarbodiimide were combined in 15 ml of cellosolve. (For the radioactively labeled compound before
addition to the reaction mixture, 125 µCi of radioactive iodoacetic
acid was added to 0.83 mmol of unlabeled iodoacetic acid in a total of
5 ml.) The reaction was initiated by the addition of a catalytic amount
of pyridine (250 µl), and the reaction mixture was allowed to stir at
room temperature for 1.5 h. The reaction was stopped by the
addition of 3 ml of distilled water, and the mixture was centrifuged to
remove the insoluble dicyclohexylurea. The organic layer, containing
17
-IES, was lyophilized. The product was resuspended in 100 µl of
acetonitrile and was brought to a final volume of 1 ml by the addition
of distilled water.
-IES was purified by HPLC using a Varian 5000LC equipped with
a Vydac C18 column (1 × 25 cm) and a UV-100 detector. The solvent system used was H2O (Solvent A) and
acetonitrile (Solvent B). The column was equilibrated with solvent A
containing 10% solvent B. After 10 min at 10% solvent B, a linear
gradient was run to 100% B in 90 min at a flow rate of 1 ml/min. The
effluent was monitored at 275 nm and 17
-IES eluted at ~28 min. For
comparison, the starting material, 17
-estradiol-3-sulfate, elutes at
~23 min.
1 cm
1, with the concentration
determined from the specific radioactivity.
= 9.6 mM
1
cm
1) in 0.1 M potassium phosphate buffer, pH
6.5, at 25 °C according to Habig et al. (22).
-IES with Glutathione S-transferase, Isozyme
1-1--
Glutathione S-transferase (0.2 mg/ml, 7.8 µM enzyme subunits) was incubated in 0.1 M
potassium phosphate buffer, pH 7.0, at 37 °C with various
concentrations of 17
-IES. Control enzyme samples were incubated
under the same conditions but without 17
-IES. At various time
points, an aliquot was removed from the incubation mixture, diluted,
and assayed (30 µl) for residual activity.
-IES into Glutathione
S-Transferase--
Glutathione S-transferase (0.2 mg/ml)
was incubated with 500 µM [14C]17
-IES at
pH 7.0 under standard reaction conditions. Aliquots were withdrawn at
various times, and excess reagent was removed by the gel centrifugation
method using two successive Sephadex G-50 columns (5 ml) equilibrated
with 0.1 M potassium phosphate buffer, pH 7.5 (23). The
protein concentration in the filtrate was determined using the Bio-Rad
protein assay, based on the Bradford method, using a Bio-Rad 2550 RIA
plate reader with a 600-nm filter (24). Unmodified GST 1-1 was used to
generate the standard concentration curve. The amount of reagent
present was determined by radioactivity using a Packard 1500 Liquid
scintillation counter. Incorporation was expressed as mol 17
-IES/mol
of enzyme subunit.
-IES at pH 7.0 under standard reaction
conditions for 3 h, at which time the enzyme was maximally
inactivated. Excess reagent was removed as described above. Solid
guanidine HCl was added to make a 5 M guanidine-HCl
solution and was incubated for 1 h at 37 °C to denature the
protein, followed by treatment with 10 mM
N-ethylmaleimide at 25 °C for 30 min to block free
cysteine residues. The solution was then dialyzed against 6 liters of
10 mM ammonium bicarbonate, pH 8, at 4 °C with one
change for a total of 18 h, after which the sample was lyophilized.
-IES
was constructed using the Builder module. Docking of 17
-IES was done manually based on the energy minimized structure of
17
-estradiol-3,17-disulfate docked into isozyme 1-1 (15).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-IES--
Incubation of rat GST 1-1 (0.2 mg/ml, 7.8 µM
enzyme subunits), with 300 µM 17
-IES, when assayed
with 1-chloro-2,4 dinitrobenzene, results in a
time-dependent loss of enzyme activity that reaches a limit
of 60% of the original activity, as is illustrated in Fig.
2A. After 180 min, excess
reagent was removed, and a second addition of 300 µM
17
-IES was added; no further decrease in activity occurred. Because
the activity levels off at 60% at long incubation times and over a
range of 17
-IES concentrations, the data were calculated using 60%
as the end point (Fig. 2B). Control enzyme incubated under
the same conditions but with no reagent present shows no loss of
activity. The kobs for inactivation was
calculated from the slope of ln([Et
E
]/[E0
E
]) versus time where
Et is the enzyme activity at time t, E0 is the original enzyme activity, and
E
is the enzyme activity at long times, which
is equal to 0.6 (E0). The reaction obeys
pseudo-first order kinetics with a rate constant of 0.0125 min
1 (Fig. 2B).
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Fig. 2.
Inactivation of glutathione
S-transferase, isozyme 1-1, by
17 -IES. A solution of 0.2 mg/ml GST 1-1 was incubated with 300 µM 17
-IES in 0.1 M
potassium phosphate buffer, pH 6.5, at 37 °C. Activity was measured
using the substrates 1-chloro-2,4-dinitrobenzene and glutathione, as
described under "Experimental Procedures." A, semilog
plot of enzyme activity at time t (Et)
versus time. B, semilogarithmic plot of [ Et
E
]/[E0
E
] versus time, where
E0 is the original enzyme activity, and
E
is the enzyme activity at long times,
which = 0.6 (E0). The apparent rate
constant (kobs) determined from this graph was
0.0125 min
1.
-IES as described above, to determine
the rate of inactivation at various reagent concentrations (Fig.
3). The apparent rate constant
kobs exhibits a nonlinear dependence on reagent
concentration. This type of curve is typical of an affinity label,
suggesting that a reversible enzyme-reagent complex is formed prior to
the irreversible modifcation of the enzyme (26). The curve can be
described by the equation kobs = kmax/(1 + KI/[17
-IES]), where KI is the apparent dissociation constant of
the enzyme-reagent complex, and kmax is the
maximum rate of inactivation at saturating concentrations of the
reagent. A least squares fit of the observed data yields
KI = 71.4 µM and
kmax = 0.0133 min
1.
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Fig. 3.
Concentration dependence of
kobs for the inactivation of glutathione
S-transferase by 17 -IES.
GST (0.2 mg/ml) was incubated with a range of concentrations of
17
-IES under the same conditions as Fig. 2. At each concentration
kobs was calculated as illustrated in Fig.
2B, with E
= 0.6 (E0). The points are experimental and the line
is the theoretical fit to kobs = kmax/(1 + (KI/[17
-IES])). A least squares fit of the data
yields KI = 71.4 µM and
kmax = 0.0133 min
1.
-IES--
Various ligand analogues were added to the reaction
mixture to determine whether they could protect against the
inactivation of the enzyme by 100 µM 17
-IES. The
results, given in Table I, are expressed
as k+L/k
L, where
k+L is the rate constant for inactivation in the
presence of a particular ligand, and k
L is the
rate constant for inactivation in the absence of a particular ligand.
Glutathione derivatives (Table I, lines 2 and 3) offer some protection,
with the protective effect increasing with an increase in alkyl chain
length. The 5 mM concentrations used are sufficient to
saturate the glutathione site, yet
k+L/k
L does not
decrease below 0.37. These results suggest that the target site of
17
-IES is near the glutathione site but distinct from it.
Electrophilic substrates, such as
5-androstene-3,17-dione (Table I, line 4), do not
provide any protection. In contrast, including steroid sulfates, such
as 17
-estradiol-3,17-disulfate, cause a striking decrease in the
observed inactivation rate constant (lines 5-10). Because these
steroid sulfates are known to bind at a nonsubstrate steroid site (15),
the results indicate that 17
-IES is reacting within this
nonsubstrate steroid binding site.
Effects of enzyme ligands on the inactivation of glutathione
S-transferase by 300 µM 17-IES
-IES into GST 1-1--
GST 1-1 (0.2 mg/ml) was incubated with 300 µM
[14C]17
-IES. A time-dependent
incorporation of [14C]17
-IES was observed concomitant
with the decrease in enzyme activity. A plot of the percentage of
maximum inactivation versus net incorporation (Fig.
4) extrapolates to ~0.5 mol of
14C-labeled reagent incorporated per mol of enzyme subunit
or 1 mol/enzyme dimer at 100% of maximum inactivation.
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Fig. 4.
Incorporation of 14C-labeled
17 -IES into GST isozyme 1-1, as a function of
the percentage of maximum inactivation. Extrapolation to the
maximum inactivation of the enzyme reveals an incorporation of about
0.46 mol reagent/mol enzyme subunit.
-IES Modified GST 1-1--
Maximally inactivated GST 1-1 was
prepared and digested with chymotrypsin. The digest was fractionated by
HPLC using a reverse-phase column (C18) equilibrated with
0.1% trifluoroacetic acid and an acetonitrile gradient (Fig.
5). One radioactive peptide peak was observed on HPLC. Because the ester linkage of 17
-IES (Fig. 1) was
hydrolyzed before the digest was applied to HPLC, the steroid moiety
was removed, and the peptide is expected to be labeled with the
radioactive carboxymethyl group. The fractions corresponding to this
peak were pooled, lyophilized, and subjected to gas phase amino acid
sequencing. The results are shown in Table
II. The sequence Glu-Xaa-Ile-Arg-Trp
corresponds to residues 16-20 in the known amino acid sequence. None
of the common phenylthiohydantoin derivatives was detected in cycle 2;
instead, there was a peak with a retention time between that of
phenylthiohydantoin-Ser and phenylthiohydantoin-Asn. This peak
corresponds to that of a phenylthiohydantoin-carboxymethylcysteine
standard, indicating that a Cys in this position had been modified.
Thus, Cys17 of GST1-1 is the amino acid target of
17
-IES.
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Fig. 5.
Fractionation by HPLC of chymotryptic digest
of [14C] 17 -IES modified
gluathione S-transferase.
Representative sequence of modified peptide (Peak I) isolated from the
chymotryptic digest of 14C-labeled glutathione S-transferase
1-1, as illustrated by Fig. 5
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Iodoacetoxy-estradiol-3-sulfate acts as an affinity label of
rat liver glutathione S-transferase isozyme 1-1. Upon
incubation of the enzyme with 17
-IES, a time-dependent
loss of activity is observed, yielding a maximum loss of 40% of the
original activity. The rate of inactivation exhibits nonlinear
dependence on reagent concentration, as is typical of an affinity
label, for which an enzyme-reagent complex forms prior to irreversible
modification. Partial protection against inactivation is provided by
glutathione derivatives; long chain derivatives, such as
S-hexylglutathione, provide more protection than do shorter
chain derivatives, like S-methylglutathione, indicating that
17
-IES is binding in a site close to the glutathione site but not
within the site. Electrophilic substrate analogues, such as
5-androstene-3,17-dione, do not offer any protection,
demonstrating that 17
-IES does not bind within the electrophilic
substrate site. Steroid sulfates are most effective in protecting
against inactivation of GST, 1-1, with 17
-estradiol-3,17-disulfate
providing complete protection. These results indicate that 17
-IES is
binding and reacting within the nonsubstrate steroid binding site.
-IES/enzyme dimer, and
Cys17 is the only amino acid that is modified. In previous
work, based on the crystal structure of glutathione
S-transferase from the parasitic worm Schistosoma
japonica in complex with praziquantel, an anti-schistosomal drug
bound in the cleft between the subunits, only 1 mol of praziquantel is
bound per mol of enzyme dimer (27). Photoaffinity labeling of rat liver
GST 1-1 by glutathionyl S-[4-(succinimidyl)-benzophenone] also results in one subunit being modified (28). Other precedence for
binding only 1 mol reagent/mol enzyme dimer comes from work with large
conjugation products, such as
S-[[(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)-carbamoyl]methyl]glutathione (29), and the aflatoxin glutathione conjugate,
8,9-dihydro-8-(S-glutathionyl)-9-hydroxyl-aflatoxin, which bind to
class glutathione S-transferases with a stoichiometry of 1 mol/mol dimer (25).
-IES results in the
loss of only 40% of activity; it is likely that the unmodified subunit
retains full activity, whereas the other subunit with modified
Cys17 is 80% inactive. Incorporation of 17
-IES on one
subunit apparently prevents a second molecule from binding to and
reacting with the other subunit, but, in contrast to the previous
examples, this does not cause complete inactivation of both subunits.
The 17
-IES reacts at the steroid site, which is distinct from the
active site, and thus there is still some residual activity in the
modified subunit, whereas the catalytic site on the other subunit
functions independently and is completely active. These results
indicate that the observation of apparent cooperativity between the
subunits of glutathione S-transferase depends on the
particular binding site that is being examined.
-estradiol-3,17-disulfate bound to GST 1-1 and the
assumptions that the iodoacetoxy group of the 17
-IES must be close
to the sulfhydryl group of Cys17 as well as in an
orientation to modify only one subunit. The structure shown in Fig.
6 meets these requirements.
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Fig. 6.
Homology model of rat GST 1-1 complexed with
17 -IES, constructed as described under
"Experimental Procedures." The reactive iodoacetoxy
group is about 3.4 Å from the sulfhydryl group of CysA17,
and the sulfate group is about 3.1 Å from the guanidino group of
Arg14.
In the proposed model, there is 1 mol of 17-IES bound in the cleft
between the subunits of the enzyme. The reactive iodoacetoxy group is
about 3.4 Å from the sulfhydryl group of CysA17. This
orientation prevents a second molecule from reacting at Cys17 on subunit B. 17
-IES appears to bind more
specifically than does 3
-(iodoacetoxy)dehydroisoandrosterone, which
modified both Cys17 and Cys111 (15). In the
case of 3
-IDA, reaction at the two sites were mutually exclusive,
i.e. reaction with Cys17 on one subunit excludes
binding and reaction with Cys17 on the other subunit. We
now propose that the more specific reaction of 17
-IES with only
Cys17 is due to an interaction between the sulfate group of
17
-IES and the guanidino group of Arg14; this
interaction would orient the reagent within the binding cleft. Based on
the model, the charged sulfate group of 17
-IES is about 3.1 Å from
the guanidino group of Arg14.
In summary, 17-IES functions as an affinity label of the
nonsubstrate steroid site of rat liver glutathione
S-transferase, isozyme 1-1. Upon incubation with 17
-IES,
the enzyme loses 40% of its activity, incorporates about 0.5 mol of
reagent/enzyme subunit, and is modified only at Cys17.
Protection against inactivation by 17
-IES is best provided by
steroid sulfates, such as 17
-estradiol-3,17-disulfate, indicating that Cys17 is within the nonsubstrate steroid binding site
of the enzyme and that its binding is more specific than that of
3
-IDA because of the interaction of the sulfate group with the side
chain of Arg14. Based on analysis of molecular models, this
nonsubstrate site is located within the cleft between the two subunits
of the enzyme.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Yu-Chu Huang for obtaining the peptide sequences.
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FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grants RO1 CA 66561 and T32 GM 08550 (to M. A. V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
302-831-2973; Fax: 302-831-6335; E-mail:
rfcolman@chem.udel.edu.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M008212200
2 Glutathione S-transferase, isozyme 1-1, is designated as the rGSTA1,2 isozyme in the nomenclature of Hayes and Pulford (5).
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ABBREVIATIONS |
---|
The abbreviations used are:
GST, glutathione
S-transferase;
3-IDA, 3
-(iodoacetoxy)dehydroisoandrosterone;
17
-IES, 17
-iodoacetoxy-estradiol-3-sulfate;
HPLC, high pressure liquid
chromatography.
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REFERENCES |
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