From the Department of Biochemistry, Albert Einstein
College of Medicine, Bronx, New York 10461 and the
§ Department of Pharmacology and Toxicology, University of
Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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Estrogen sulfotransferase (EST) catalyzes the
transfer of the sulfuryl group from 3'-phosphoadenosine
5'-phosphosulfate (PAPS) to 17-estradiol (E2). The
sulfation of E2 prevents it from binding to, and thereby
activating, the estrogen receptor. The regulation of EST appears to be
causally linked to tumorigenesis in the breast and endometrium. In this
study, recombinant human EST is characterized, and the catalytic
mechanism of the transfer reaction is investigated in ligand binding
and initial rate experiments. The native enzyme is a dimer of 35-kDa
subunits. The apparent equilibrium constant for transfer to
E2 is (4.5 ± 0.2) × 103 at pH 6.3 and
T = 25 ± 2 °C. Initial rate studies provide
the kinetic constants for the reaction and suggest a sequential
mechanism. E2 is a partial substrate inhibitor
(Ki = 80 ± 5 nM). The binding of
two E2 per EST subunit suggests that the partial inhibition
occurs through binding at an allosteric site. In addition to providing
the dissociation constants for the ligand-enzyme complexes, binding
studies demonstrate that each substrate binds independently to the
enzyme and that both the E·PAP·E2S and
E·PAP·E2 dead-end complexes form. These
results strongly suggest a Random Bi Bi mechanism with two dead-end
complexes.
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INTRODUCTION |
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Transferring the sulfuryl group
(-SO3) from activated sulfate, or
PAPS,1 to a given metabolic
recipient typically switches "on" or "off" or otherwise
modifies the function of that metabolite (1-6). The extent of
sulfation of a given metabolite is determined by the regulated
expression of two enzyme classes: the sulfotransferases, which transfer
the sulfuryl group, and the sulfatases, which remove it. PAPS appears
to be the sole sulfuryl group donor in metabolism. The chemical
potential associated with the phosphoric-sulfuric acid anhydride bond
of PAPS is remarkably high
(
Ghydrolysis0' ~
19
kcal/mol) (7). Thus, the sulfuryl moiety is well positioned energetically for facile and favorable transfer to its metabolic recipients. The chemical and regulatory parallels between
sulfation/desulfation and phosphorylation/dephosphorylation are quite
strong, yet relatively little is know about the chemistry and
enzymology of sulfuryl transfer.
The transcriptional activity of the estrogen receptor (ER) is regulated
by sulfation/desulfation at the 3-hydroxyl position of 17-estradiol
(E2). The binding of E2 to the ER, located in the nuclear membrane, elicits a complex cellular response that is
rooted in the transcriptional activity of the ER·E2
complex. E2 binds tightly to the ER (Kd ~ 1 nM) (8, 9); E2S, on the other hand, binds
weakly, if at all (10). Thus, the ER-binding activity of estrogen (and,
in turn, ER activation) is regulated by E2 sulfation. The
sulfation of E2 (Reaction 1),
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Human liver estrogen sulfotransferase (EST) has been cloned and expressed in Escherichia coli K1 (13). In this paper, recombinant EST purified to homogeneity from E. coli is physically characterized. The equilibrium constant for Reaction 1 is determined, and the catalytic mechanism of EST is evaluated in initial rate and ligand binding studies.
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EXPERIMENTAL PROCEDURES |
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Materials-- The buffers, salts, enzymes, and reagents, unless specified otherwise, were of the highest grades available from Sigma. [2,4,6,7-3H]Estradiol (85 Ci/mmol) was purchased from NEN Life Science Products. Adenosine 3',5'-[5'-32P]bisphosphate (3000 Ci/mmol) was purchased from ICN Pharmaceuticals. PAPS was purchased from Professor S. Singer (University of Dayton, Dayton, OH). Factor Xa protease was purchased from Enzyme Research Labs. The Bradford assay reagents were purchased from Bio-Rad. The Superdex 200 column was purchased from Amersham Pharmacia Biotech. Amylose resin was obtained from New England Biolabs Inc.
Purification of Estrogen Sulfotransferase--
Competent
XL1-Blue (14) cells were transformed with the estrogen sulfotransferase
expression vector pMBP-hEST-1 (13) and immediately used to inoculate a
100-ml culture of LB medium (15) containing ampicillin at 50 µg/ml.
The pMBP-hEST-1 vector directs the synthesis of a maltose-binding
protein-human EST-1 fusion protein, which can be purified by amylose
affinity chromatography (13). The culture was incubated overnight at
37 °C and then used to inoculate 10 liters of LB medium to
A600 = 0.06. At A600 = 0.6, isopropyl--D-thiogalactopyranoside was added to 350 µM. 3.5 h later, the cells were pelleted at
3500 × g for 25 min, suspended in 500 ml of buffer
containing 20 mM Tris-Cl (pH 7.4), 0.20 M KCl,
and 5 mM
-mercaptoethanol, and frozen at
70 °C. All
of the purification steps were performed at 4 °C. The cells were thawed, and 1 liter of 4 °C lysis buffer (115 mM Tris-Cl
(pH 8.0), 0.375 M sucrose, 0.375 mM EDTA, and
0.03 mg/ml lysozyme) was added. 20 min later, the cells were pelleted
at 3500 × g for 25 min. The cell pellet was suspended
in sonication buffer (5.0 mM KPO4 (pH 7.4), 1.5 mM DTT, and 57 µM phenylmethylsulfonyl
fluoride) and sonicated. The cellular debris was pelleted at
27,000 × g for 60 min, and the concentration of
protein, determined by the method of Bradford (16), was adjusted to 3.0 mg/ml using 5.0 mM KPO4 (pH 7.4).
Native Molecular Mass-- The native molecular mass of EST was determined by size-exclusion chromatography using a Superdex 200 (XK 27/70) column. The column was equilibrated and run in 50 mM K+-Hepes (pH 8.0) at 4 °C. Bio-Rad gel filtration standards were used to calibrate the column. The apparent native molecular mass of EST was 62 ± 2.3 kDa.
Extinction Coefficient--
The extinction coefficient of EST
was determined gravimetrically at 280 nm, the max for
the enzyme. EST was dialyzed at 4 °C for 8 h and then overnight
against 10 mM NaPO4 (pH 6.3), 1.0 mM KCl, and 0.10 mM DTT. The absorbance of the
dialyzed EST was determined in triplicate at 280 nm in a masked 1-cm
path length cuvette. Dialyzed enzyme or dialysis buffer (200 ± 1.6 µl) was added to an aluminum weigh boat and dried under vacuum
and over P2O5 to a constant weight (<40 h).
Triplicate samples of the dialyzed enzyme and buffer were each weighed
three times. The absorbance at 280 nm was divided by the concentration
of enzyme to calculate the extinction coefficient:
280* = 1.7 ± 0.1 A280 × (mg/ml)
1 × cm
1.
Equilibrium Constant-- The equilibrium constant for the EST reaction was determined at pH 6.3 and T = 22 ± 2 °C. The measurements were performed in duplicate at each of the following three sets of initial concentrations of E2S, [5'-32P]PAP, and EST, respectively: 100 µM, 240 nM, and 8.0 nM; 200 µM, 240 nM, and 8.0 nM; and 500 µM, 630 nM, and 32 nM. The progress curve for each reaction was determined, and the equilibrium constant was calculated from the reactant concentrations in the stationary phase of the progress curve. The buffer was the same as that used in the initial rate experiments. The concentration of product formed in each reaction was at least 10 times the enzyme active-site concentration. The equilibrium constant was (4.5 ± 0.2) × 103.
Divalent Cation Activation-- The initial rate of the forward reaction was studied as a function of MgCl2 concentration. The assays were performed as described below under "Initial Rate Studies of the Forward Reaction." The conditions of these experiments were as follows: 1.0 nM EST, 10 nM E2, 6.0 µM PAPS, 50 mM KPO4 (pH 6.3), 1.0 mM DTT, and 10% (v/v) glycerol at T = 25 ± 2 °C. The MgCl2 concentration was varied in 2.0 mM increments from 0 to 11 mM. EDTA was added to 2.0 mM in the experiments at zero MgCl2 to ensure that the observed activity was not caused by trace divalent cations in the buffer.
Initial Rate Studies of the Forward Reaction-- The reactions were initiated by adding 50 µl of enzyme to 200 µl of a buffered solution containing varying concentrations of E2 and PAPS. The buffer used in these experiments contained 50 mM KPO4 (pH 6.3), 7.0 mM MgCl2, 1.0 mM DTT, and 10% (v/v) glycerol. The reaction was quenched by the addition of 950 µl of 10 mM KOH. (Controls were run to ensure that the KOH did not hydrolyze the E2S produced.) 5.0 ml of CHCl3 was then added, and the solution was vortexed for 15 s. 800 µl of the aqueous phase was removed and counted. Velocities were determined in duplicate under each of the 16 conditions defined by a 4 × 4 matrix of substrate concentrations. The concentration of EST was 0.10 nM in all of the experiments. The concentrations of PAPS (12, 18, 34, and 300 nM) ranged from 0.2 to 5 times its Km. The concentrations of E2 (2.0, 2.8, 4.8, and 15.5 nM) ranged from 0.4 to 3 times its Km. Partial substrate inhibition by E2 is negligible at these E2 concentrations. The velocities were measured under initial rate conditions since the consumption of the concentration-limiting substrate was <7% of its end point in all cases. The data were statistically fit to the sequen model using the program developed by Cleland (17).
Initial Rate Studies of the Reverse Reaction-- 5.0 µl of EST (4.0 nM) was added to 15 µl of solution containing PAP at varying concentrations and E2S at 250 µM. The reactions were quenched by the addition of 4.0 µl of 0.10 M KOH (final pH 10.0). 9.0 µl of solution was spotted onto polyethyleneimine-fluorescamine TLC plates. The radiolabeled reactants were separated using a LiCl (0.50 M) and HCO2H (2.0 M) mobile phase and quantitated using an AMBIS two-dimensional radioactivity detector. The experiments were performed at 25 ± 2 °C. The data were statistically fit to the hyper model using the weighted least-squares program of Cleland (17).
Substrate Inhibition by E2-- These initial rate assays were performed as described above for the forward reaction. The concentrations of EST and PAPS were 1.0 and 600 nM, respectively. To determine whether inhibition was caused by changes in kcat or the Km for PAPS, the rates at the highest concentrations of E2 (i.e. the plateau region of the curve shown in Fig. 2) were performed at a 10 times higher concentration of PAPS (6.0 µM). Each velocity was determined in duplicate. The data were fit using a partial substrate inhibition model (see "Results and Discussion") with the program Kaleidograph, which uses the Marquardt-Levenberg minimization algorithm.
Fluorescence Titrations--
The EST fluorescence excitation and
emission wavelength maxima are 275 and 340 nm, respectively. To avoid
possible inner filter effects caused by the absorption of PAP and PAPS,
a 285-nm excitation wavelength was used in all of the experiments. The
highest nucleotide concentration used in the titrations (5.5 µM) corresponds to an absorbance of 4.7 × 104. This absorbance is ~100-fold below the threshold
where inner filtering begins to influence intensity measurements (18).
The emitted light was detected at 340 nm in all experiments except those involving E2, which is weakly fluorescent when
excited at 285 nm. In experiments involving E2, the
emission wavelength was set at 360 nm. At this wavelength, the emission
from E2 is negligibly low (<0.2% of the EST emission at
an equivalent concentration). The solutions used in the titrations were
thermally equilibrated and maintained at 25 ± 2 °C during the
experiment. The buffer (50 mM KPO4 (pH 6.3),
1.0 mM DTT, 7.0 mM MgCl2, and 10%
(v/v) glycerol) was filtered using Gelman 0.2-µm Acrodiscs. The
fluorometer used in these studies was a Perkin-Elmer Model LS-5B; the
entrance and exit slit widths were set at 10 nm. The titration data
were fit to a single-site binding model using the Sigma Plot program, which employs the Marquardt-Levenberg fitting algorithm. The
single-site binding model is described by a second order polynomial.
The data were fit to the appropriate root of this polynomial to obtain the best fit binding constants.
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RESULTS AND DISCUSSION |
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Native Molecular Mass and Extinction Coefficient of EST--
The
apparent native molecular mass of EST, determined by size-exclusion
chromatography (see "Experimental Procedures"), is 62 (±2.3) × 103 Da. The subunit molecular mass, predicted from the DNA
sequence of the EST coding region, is 35,123 kDa (13). Thus, the native enzyme appears to be a dimer. The extinction coefficient of EST, determined gravimetrically, is 1.7 ± 0.1 A280 × (mg/ml)1 × cm
1 (see "Experimental Procedures"). The
experimentally determined
280* is
identical to that calculated for EST from its amino acid composition and the
280* of Trp and Tyr (19).
Stabilizing EST Activity--
In the absence of glycerol, the
activity (i.e. the initial rate of PAP and E2S
synthesis) of EST is stable for several days at 70 °C, and the
half-life of the activity is ~2 h at 25 °C. Glycerol (10%, v/v)
prevents detectable deterioration of the activity over 3 h at
25 °C. The addition of E2 to EST in glycerol causes a
rapid loss of activity (t1/2 ~ 30 min). This
E2-induced inactivation was prevented by the addition of
DTT. At 1.0 mM DTT, the activity was not affected at a
saturating concentration of E2 over 4 h at 25 °C.
Thus, it appears that the binding of E2 potentiates an
inactivating oxidation reaction that is suppressed by DTT.
Optimizing Turnover-- Most enzyme-catalyzed transfer reactions involving nucleotides require divalent cations. In these cases, the cations are often directly bound to the polyphosphate chain of the nucleotide. The mechanism of this activation appears to be due predominantly to the entropy reduction associated with the positioning of functional groups for reaction (20). It is interesting that while sulfotransferases catalyze transfer reactions that, in many ways, resemble phosphoryl transfer reactions, they do not require divalent cations for activity. They are, however, activated by divalent cations. A plot of the initial rate of the forward EST reaction versus [MgCl2] is bell-shaped with a maximum at 7.0 mM MgCl2 (see "Experimental Procedures"). The initial rate at zero MgCl2 and 2.0 mM EDTA is 0.18 times the initial rate at 7.0 mM MgCl2. The buffers used in the mechanism studies described in this paper contained MgCl2 at 7.0 mM.
To determine an optimum pH for the EST mechanism studies, the initial rate of the forward reaction was studied as a function of pH at subsaturating E2 and saturating PAPS concentrations. This condition maximizes turnover with respect to both the Km for E2 and kcat. The assay protocol was as described under "Initial Rate Studies of the Forward Reaction", except for the following changes. EST at 0.50 nM and PAPS at 6.0 µM were used, and the pH of the buffer (50 mM KPO4) was varied in 0.4 pH unit increments between 5.4 and 7.4, inclusive, by mixing dibasic and monobasic solutions of KPO4. The pH rate profile was bell-shaped with a maximum initial rate at pH 6.3. The recent structure of mouse testis estrogen sulfotransferase implicates His-108 as a general base that abstracts a proton from the 3-hydroxyl group of E2, thereby activating it for attack (21). Further pH rate studies will help to determine whether this residue contributes to the pH rate dependence of the EST-catalyzed reaction.Equilibrium Constant--
To evaluate the energetics associated
with transferring the sulfuryl group between PAPS and E2
and to aid in the design of initial rate experiments, the equilibrium
constant for the EST reaction was determined at pH 6.3 and
T = 25 ± 2 °C (the conditions of the initial
rate studies). The equilibrium constants were calculated from reactant
concentrations in the stationary phases of reaction progress curves
constructed in duplicate at three different sets of E2S and
PAP concentrations (see "Experimental Procedures"). Controls were
run to ensure that the EST activity did not change during the
experiments. The equilibrium constant is (4.5 ± 0.2) × 103. The G0 associated with this
Keq is
5.0 kcal/mol. It should be emphasized this apparent equilibrium constant does not explicitly include the
proton and divalent cation dependences.
Initial Rate Study of the Forward Reaction--
To determine the
kinetic constants for the forward reaction and to obtain a preliminary
assessment of the order of substrate binding, a classical initial rate
study of the forward reaction was performed. The initial rate was
determined as a function of both E2 and PAPS concentrations
(see "Experimental Procedures"). The results of this study are
shown in Fig. 1. The pattern of the data
is indicative of a sequential mechanism (one in which both substrates
must bind to the enzyme before product is released). However, it does
not rule out a ping-pong mechanism with an unstable enzyme
intermediate. An equilibrium ordered mechanism is ruled out by the fact
that the lines through the data of the 1/V versus 1/[E2] and 1/V versus 1/[PAPS] (not shown)
plots do not intersect on the 1/V axis (22). The kinetic
constants obtained from this study are compiled in Table
I. kcat (1.3 ± 0.08 s1) and the Km for PAPS
(59 ± 13 nM) are similar to those measured for other
sulfotransferases (23, 24). The Km for
E2 is comparable to the in vivo concentration of
E2, ~1 nM (25), suggesting that the enzyme is
optimized to perform at the physiological concentration of
E2.
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Initial Rate Study of the Reverse Reaction--
The
Km for PAP and kcat for the
reverse reaction were determined in an initial rate study at a
saturating (920 × Kd) concentration of
E2S (250 µM). Controls were run to ensure
that E2S did not inhibit the velocity at this
concentration. The Km for PAP was 38 ± 0.8 nM, and kcat was 0.16 ± 0.0013 min1 (Table I). The experimental protocol is described
under "Experimental Procedures." Given the technical obstacles
associated with the unfavorable equilibrium constant for the reverse
reaction and the relatively high Km for
E2S, the order of substrate addition for the reverse
reaction was determined with the equilibrium binding studies described
below.
Partial Substrate Inhibition by E2--
The initial
rate data shown in Fig. 2 demonstrate
that E2 inhibits the forward reaction. The fact that the
velocity decreases to a plateau, rather than to zero, means that one or
more of the kinetic parameters for the reaction are being titrated from
one value to another as E2 adds to the enzyme. The
inhibition experiment (Fig. 2, ) was performed at a fixed
near-saturating concentration of PAPS (600 nM, 10 × Kd). If the inhibition were due solely to an
increase in the Km for PAPS, causing the concentration of the reactive form(s) of the enzyme to decrease, increasing the concentration of PAPS would drive the initial rates, at
inhibitory concentrations of E2, back to the uninhibited
levels. This, in fact, does not occur. Increasing the concentration of PAPS 10-fold (Fig. 2,
) had no significant effect on the initial rates at high E2 concentrations. Thus, it is
kcat that is affected by the binding of
E2 in these experiments.
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Equilibrium Binding Studies--
The excitation and emission
wavelength maxima for EST are 275 and 340 nm, respectively. The fine
structure and max of the emission spectrum do not change
significantly when substrates bind to EST; however, the intensity
decreases 30-50% depending on the ligand. These
ligand-dependent decreases in the quantum yield of EST
provide excellent experimental handles to determine both the
equilibrium constants and stoichiometry of the enzyme-ligand interactions.
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Conclusions-- Initial rate and ligand binding experiments have been used to investigate the catalytic mechanism of EST. The kinetic parameters for the mechanism were determined from the initial rate studies, which also suggested that the mechanism is sequential. Ligand binding studies were used to determine the equilibrium constants and stoichiometries of the enzyme-substrate interactions. The binding studies demonstrated that each of the substrates can bind independently to the enzyme and that two dead-end complexes can form. These results strongly suggest a Random Bi Bi mechanism with two dead-end complexes. The initial rate experiments revealed that E2 is a partial substrate inhibitor of the reaction with a Ki of 80 ± 5 nM. The mechanism of the inhibition is partially delineated by the stoichiometry studies, which show that the enzyme contains two E2-binding sites/catalytic subunit, suggesting that the enzyme harbors an allosteric E2-binding site.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM54469 (to T. S. L.) and GM38953 (to C. N. F.).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: Dept. of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2857; Fax: 718-430-8565.
1
The abbreviations used are: PAPS,
3'-phosphoadenosine 5'-phosphosulfate; PAP, adenosine
3',5'-diphosphate; ER, estrogen receptor; E2,
17-estradiol; E2S, 17
-estradiol 3-sulfate; EST,
estrogen sulfotransferase; DTT, dithiothreitol.
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REFERENCES |
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