From the Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden
Received for publication, October 6, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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Human glutathione transferase (GST) A1-1
efficiently catalyzes the isomerization of
The biosynthesis of hormones such as testosterone,
progesterone, and corticosteroids includes 35-androstene-3,17-dione (AD) into
4-androstene-3,17-dione. High activity requires
glutathione, but enzymatic catalysis occurs also in the absence of this
cofactor. Glutathione alone shows a limited catalytic effect.
S-Alkylglutathione derivatives do not promote the reaction,
and the pH dependence of the isomerization indicates that the
glutathione thiolate serves as a base in the catalytic mechanism.
Mutation of the active-site Tyr9 into Phe significantly
decreases the steady-state kinetic parameters, alters their pH
dependence, and increases the pKa value of the
enzyme-bound glutathione thiol. Thus, Tyr9 promotes the
reaction via its phenolic hydroxyl group in protonated form. GST A2-2
has a catalytic efficiency with AD 100-fold lower than the homologous
GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less
active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than
GST A2-2 and GST A4-4, both having a glutathione cofactor and an
active-site Tyr9 residue. The active sites of GST A2-2 and
GST A1-1 differ by only four amino acid residues, suggesting that
proper orientation of AD in relation to the thiolate of glutathione is
crucial for high catalytic efficiency in the isomerization reaction.
The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3
-hydroxysteroid dehydrogenase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxysteroid
oxidation followed by a
5 to
4
isomerization of the 3-ketosteroid
product. In bacteria, such as
Pseudomonas species, the two consecutive steps are
catalyzed by two distinct enzymes, a 3
-hydroxysteroid dehydrogenase
and a 3-ketosteroid isomerase (1). Mammalian tissues contain
3
-hydroxysteroid dehydrogenases, which have been found to catalyze
also the isomerase reaction (2-4). Extending the studies of the
isomerization (Fig. 1) of
5-androstene-3,17-dione
(AD)1 to mammalian tissues,
Benson and Talalay (5) identified a glutathione
(GSH)-dependent enzyme in rat liver, which was later identified (6) with a major glutathione transferase (GST). The multiple
forms of GST are divided into different classes, based on their
primary structures. The different classes of soluble mammalian GSTs
have been denoted2 Alpha, Mu,
Pi (8), Theta (9), Sigma (10), Kappa (11), Zeta (12), and Omega (13).
The proteins are dimers, each subunit containing a largely conserved
GSH-binding site (G-site) and a more promiscuous site (H-site) for the
second, electrophilic, substrate. In GST A1-1 about 16 of the 25 residues in the cavity of the active center are regarded as H-site
residues and the remaining 9 as G-site residues (14). The active-site
Tyr9 residue plays an important role in stabilizing and
orienting the thiolate form of GSH in the conjugation reactions
catalyzed by GST A1-1 (14, 15). A crevice between the two subunits of soluble GSTs may serve as an additional binding pocket distinct from
the active site (16, 17).
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Fig. 1.
The isomerization of
5-androstene-3,17-dione catalyzed by
GST A1-1. The product,
4-androstene-3,17-dione,
absorbs strongly at 248 nm, while the substrate does not.
Extensive studies have been made of the role of the GSTs in detoxication reactions between GSH and different electrophiles both in normal cells (18-20) and in neoplastic cells where they possibly give rise to multiple drug resistance in tumors subjected to chemotherapy (21, 22). Structural determinants governing protein folding and stability of GSTs have also been identified (23). The three-dimensional structures (24, 25) and the catalytic functions (20) of naturally occurring GSTs have been intensively studied, and their redesign into enzymes with novel properties has been accomplished by means of protein engineering (26, 27).
However, the GST-catalyzed isomerization of AD has not been subjected
to detailed mechanistic studies despite its theoretical interest and
its possible physiological significance. This GST reaction complements
the activity of the bifunctional 3-hydroxysteroid dehydrogenase/
5
4isomerase (3
-HSD/Iso),
but its relative efficiency and the role of GSH in catalysis have not
previously been evaluated.
In the present investigation the isomerization of AD catalyzed by the
major human liver enzyme GST A1-1 has been studied in detail. This
abundant isoenzyme was shown to be significantly more active with AD
than the isomerase component of the bifunctional 3-HSD/Iso
previously described. Marked differences among related Alpha class GSTs
in their ability to catalyze the isomerization of AD as well as the
significance of an ionized thiol group of GSH acting as a base in the
catalytic mechanism were also demonstrated.
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EXPERIMENTAL PROCEDURES |
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Materials--
1-Chloro-2,4-dinitrobenzene (CDNB),
S-methylglutathione (GSMe), and reduced glutathione were
obtained from Sigma.
2-Chloro-3,5-dinitro-1,1,1-(trifluoromethyl)benzene (o-CF3CDNB) was obtained from Aldrich.
5-Androstene-3,17-dione was obtained from Steraloids
(Newport, RI). S-Hexylglutathione (GSHex) was provided by
Dr. Mikael Widersten of our department.
Expression and Purification of Recombinant Enzymes--
High
level expression clones of human GST A1-1, GST A1-1 mutant Y9F, GST
A2-2, and GST A4-4 were used to produce enzyme in Escherichia
coli. The GST A1-1 clones have been described in detail (15, 28).
The expression of wild-type GST A1-1 was based on the pET21 vector
(Novagen, Inc., Madison, WI) into which it was subcloned by Ann
Gustafsson.3 cDNA
encoding GST A2-2 (29) was amplified from a human hepatoma cDNA library and engineered into the pGTacEco vector (30) by Kristina Svensson in our
laboratory.4 These enzymes
were expressed and purified using HiTrap SP cation exchange as
described earlier for GST A1-1 by Gustafsson and Mannervik (31) with
one or two additional steps of cation exchange chromatography. This
procedure yielded highly pure and concentrated protein solutions as
confirmed by SDS-polyacrylamide gel electrophoresis and
spectrophotometric analysis. GST A4-4 was provided by Dr. Ina Hubatsch
(32). Extinction coefficients used for enzyme subunit concentration
determination:
= 24,700 M
1 cm
1
for GST A1-1, Y9F, and GST A2-2 (33);
= 15,900 M
1 cm
1
for GST A4-4.5
Assays of Enzyme Activity-- The assay system for the results presented in Table I, contained 50 mM Tris-HCl at pH 8.0, 1% (v/v) methanol as solvent for the substrate, 100 µM AD and, when used, GSH or GSMe at 1 mM concentration. GST A1-1 subunit concentrations were 3.65 µM in the cases with GSMe or no GSH and 7.3 nM in the case with GSH.
Specific activity measurements with CDNB (1 mM) were made in 100 mM sodium phosphate at pH 6.5 with 1 mM GSH and monitored at 340 nm. Specific activity measurements and steady-state kinetic experiments (except for the pH dependence studies) with AD (100 µM) were made in 50 mM Tris-HCl at pH 8.0 with 1 mM GSH and monitored at 248 nm.
Parameter values presented in Table II, other than the specific activities, for GST A1-1 represent 82 measurements in the ranges of 10-200 µM for AD and 25-2000 µM for GSH. The parameters of Y9F represent 40 measurements in the ranges of 10-400 µM for AD and 1-2000 µM for GSH. GST A2-2 parameters represent 39 measurements in the ranges of 50-400 µM for AD and 55-2200 µM for GSH, while GST A4-4 parameters are based on 68 measurements in the ranges of 25-400 µM for AD and 125-2000 µM for GSH.
The reactions, in which the isomerization of AD in the presence of enzyme and GSH was measured, were initiated by the addition of the enzyme.
Kinetic studies were made on a Shimadzu UV-PC2501 spectrophotometer at 30 °C. Regression analyses were made with the computer program package SIMFIT (34), and parameter values reported are given with S.D. values.
Competitive Substrate and Inhibition Experiments-- Competition experiments were performed by measuring the conjugation of GSH with the alternative substrate o-CF3CDNB at 5 mM GSH with four different concentrations of AD (0, 75, 150, and 300 µM) and o-CF3CDNB varied from 20 to 400 µM in 44 measurements. o-CF3CDNB is a CDNB analogue that is monitored the same way as CDNB but which gives improved saturation kinetics (35). Similarly, inhibition of the isomerization reaction by GSHex was measured in a series of reactions where AD was varied from 20 to 300 µM in 42 measurements at 5 mM of GSH and different concentrations of GSHex (0, 2, 4, and 8 µM). The results of these measurements are presented in Table III.
Determination of the pKa Value of the GSH Thiol-- The spectrum of free GSH in solution was obtained at different pH values, and the ionization of the thiol group was monitored by measuring the absorbance of the thiolate at 239 nm. Spectra of GSH bound to GST A1-1 and to Y9F were obtained by subtracting the spectra of free GSH at different pH values and of free enzyme at pH 6 from the spectrum of the sample. Concentration of enzyme was 9 µM, and the GSH concentration was 250 µM; measurements were done in 100 mM sodium phosphate.
pH Dependence of the Isomerization Reaction Catalyzed by GST
A1-1--
Kinetic parameters for GST A1-1 and Y9F were determined at
different pH values with 2 mM GSH, at least 80% saturating
in the crucial part of the pH range examined, and varying AD
concentrations. Parameters for GST A1-1 in the absence of GSH were also
obtained. Sodium phosphate buffers of 100 mM were used in
the pH range 4.7-8. Above pH 8 and up to 9.7, 100 mM
ethanolamine HCl was used.
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RESULTS |
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GSH Dependence of the Enzyme-catalyzed Isomerization-- In the absence of GSH and enzyme a slow but measurable conversion of 100 µM AD into its isomer was observed (Table I). GSH (1 mM) afforded a 2-fold increased reaction rate at pH 8.0. The presence of GST A1-1 under conditions similar to those used for specific activity measurements, but in the absence of GSH, increased the rate of the isomerization about 16 times at 1 µM enzyme concentration (Table I). The presence of both GST A1-1 and GSH further increased the reaction rate 240 times so that the overall rate enhancement was about 3800-fold. Replacement of GSH by 1 mM GSMe gave an enzymatic activity lower than that in the absence of GSH, demonstrating that GSMe inhibits the GST A1-1-catalyzed reaction. The nonenzymatic isomerization rate was the same in the presence and absence of GSMe.
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Optimization of Conditions for Specific Activity
Determinations--
Modifications of the original assay system
defining the specific activity (5) increased the specific activity of
GST A1-1 from ~10 to 40 µmol min1
mg
1 and brought the assay closer to
physiological conditions. With the new conditions (50 mM
Tris-HCl, pH 8.0, 1.0 mM GSH, 100 µM AD, and
1%, v/v, methanol) the blank reaction, in the absence of enzyme, was
low in comparison with the catalyzed reaction, and data were reproducible.
Steady-state Kinetics of Alpha Class GSTs with
5-Androstene-3,17-dione--
Steady-state kinetic
experiments were made in which both GSH and AD concentrations were
varied independently. In addition, specific activities with both CDNB
and AD were determined (Table II). The
data were in agreement with a random sequential two-substrate mechanism
for all GSTs investigated (cf. Fig.
2). The kinetic parameters were obtained
by fitting Equation 1 to the experimental data.
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(Eq. 1) |
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The kcat value for GST A1-1 with AD, 29 s1, is about 50 times, 110 times, and 1200 times higher than the values for Y9F, GST A2-2, and GST A4-4,
respectively. The KmGSH values range
between 15 and 230 µM, with the Y9F value being lowest
and the GST A4-4 value highest. The KmAD
values are all of the same order of magnitude except that for GST A2-2,
which is 4-10 times higher. The values for GST A1-1 and GST A4-4 are
the same within the experimental error. The reaction rate enhancement
afforded by GST A1-1, defined as the ratio of kcat/KmAD to the
rate constant of the uncatalyzed reaction was calculated as (1.3 ± 0.1)·1010
M
1.
Competing Alternative Substrate and Dead-end Inhibition--
AD
inhibited the GST A1-1-catalyzed GSH conjugation of the alternative
substrate o-CF3CDNB in a linear competitive manner (Fig.
3). Nonlinear regression analysis,
fitting Equation 2 to the experimental data, gave a
KiAD value of 81 ± 6 µM (Table III).
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(Eq. 2) |
Inhibition by GSHex of the GST A1-1-catalyzed isomerization of AD showed linear competition with respect to both AD and GSH. At a constant GSH concentration (5 mM) and varying AD concentrations, a KiGSHex value of 5.8 ± 1.0 µM was determined.
Ability of the Enzyme to Lower the GSH Thiol pKa upon
Binding--
Spectrophotometric titration of the GSH thiolate gave
different pKa values for free GSH, GSH bound to GST
A1-1, and GSH bound to Y9F (Fig. 4). Upon
binding of GSH to wild-type GST A1-1, the thiol pKa
of 9.2 is lowered by 2.5 pH units to 6.7, in agreement with the
previous estimate of pKa 6.7 (37). The mutant
Y9F only decreased the thiol pKa by 2.0 pH units to
7.2.
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pH Dependence of the Isomerization Reaction Catalyzed by
GSTA1-1--
The pH dependence of the GST A1-1-catalyzed isomerization
of AD, at saturating levels of GSH, shows two pKa
values for kcat, at pH 6.1 and 9.5, and two for
kcat/KmAD, at 6.1 and 8.7 (Fig. 5). The reaction rate of
wild-type GST A1-1 in the absence of GSH is ~100
times6 lower at pH 8.0 and
shows only one pKa value at pH 7.8 for
kcat and one at pH 8.0 for
kcat/KmAD. The
corresponding activity for Y9F with GSH is about 35 times lower than
the wild-type activity and also displays only single pKa values, for kcat at pH
8.2, and for
kcat/KmAD at pH
8.4, respectively, slightly higher than the values in the absence of
GSH. At pH 8, in the absence of GSH, and with as much as 12 µM Y9F, the reaction rate was lower than the nonenzymatic rate observed with AD in the absence of enzyme, indicating partial inhibition of the isomerization by binding of AD to the
protein.
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DISCUSSION |
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The 5-3-ketosteroid AD spontaneously rearranges to
the
4-isomer product at a slow, but indeed, measurable
rate. The presence of 1 mM GSH markedly raises this rate,
while the same concentration of GSMe gives no detectable effect as
compared with the rate observed with AD alone. Since the sulfur of GSMe
is blocked by a methyl group, this suggests that the sulfhydryl group
of GSH is important for the catalytic effect.
GST A1-1, in the absence of GSH, also increases the rate of the
reaction as compared with either the presence of AD alone or the
presence of AD together with GSH (Table I). The largest reaction rate
enhancement, ~4000-fold, is obtained when AD, GSH, and GST A1-1 are
all present (Table I). It should be noted that in tissues the enzyme
concentration may be 100-fold higher (100 µM) with a
corresponding higher rate enhancement. However, when GSMe is
substituting for GSH, the activity of GST A1-1 decreases to a level
below the rate for GST A1-1 and AD alone. It is possible that GSMe
binds to the enzyme with its methyl group entering the binding site for
AD, thereby blocking entry of the steroid. An alternative explanation
would be that the 5-3-ketosteroid transformation is
attenuated indirectly by the interaction between enzyme and GSMe. The
lack of catalytic effect of GSMe also demonstrates that GSH does not
promote the reaction simply through a ligand-induced conformational
change of the enzyme into a catalytically competent state.
The simultaneous action of GST A1-1 on AD and o-CF3CDNB, an alternative substrate more reactive than its analogue (35), CDNB, results in alternative substrate competition (Fig. 3) with an apparent KiAD value of 81 µM (Table III) similar to the KmAD value of 58 µM (Table II). The conjugation reaction is measured at pH 6.5 and the isomerization reaction at pH 8.0, but since KmAD values of the data presented in the kcat/KmAD curve (Fig. 5B) show no marked change with pH, these results suggest that the similar KiAD and KmAD values reflect the same interaction with the protein and that the alternative substrates AD and o-CF3CDNB compete for the same binding site.
GSHex is a competitive inhibitor of conjugation reactions catalyzed by GST A1-1. Thus, GSHex competes with GSH for the G-site; the hexyl group of the inhibitor binds into the H-site and obstructs the steroid from binding. This, together with the results from the alternative substrate experiments, indicates that the isomerization of AD takes place in the active center of GST A1-1 and that AD occupies the H-site. This rules out the possibility that catalysis takes place in the cleft between the two protein subunits (16, 17). The conclusion is supported by results with the homologous rat GST A1-1 showing that AD does not protect against affinity labeling with steroid derivatives that target the nonsubstrate steroid binding site (38, 39).
At saturating levels of GSH, the pH dependence profile for the GST A1-1-catalyzed isomerization of AD has a bell shape that suggests two ionizable groups to be involved in the catalysis. Since AD does not contain any protolyzable groups, the ionizations have to be ascribed the binary GSH-enzyme complex. The pKa value corresponding to the acidic limb of this curve is 6.1 for both kcat and kcat/KmAD (Fig. 5). Because the parameter kcat describes the pH dependence for the enzyme-substrate complex, while the parameter kcat/Km describes the condition where enzyme and substrate are free, the similar pKa values indicate that the binding of substrate does not affect the group responsible for the lower pKa. The thiol group of GSH bound to the wild-type enzyme has a pKa of 6.7 (Fig. 4), somewhat higher than the kinetic pKa of 6.1. Despite this apparent discrepancy the lower kinetic pKa value is ascribed to GSH, because complex mechanisms, including pH-dependent changes of rate-determining steps, may give apparent ionization constants of active-site residues (40). With the exception of Tyr9, which has a pKa value of 8.1 (41), there is no other ionizable group in the active site of GST A1-1. Thus, the data suggest that GSH is acting as a base in the isomerization reaction. For the basic limb of the pH profiles, a difference between the pKa values of the parameters kcat and kcat/KmAD was noted (9.5 and 8.7, respectively, Fig. 5). The higher value for kcat indicates that the second of the ionizable groups becomes more basic upon binding of AD, suggesting a more hydrophobic environment.
Without GSH present in the reaction of GST A1-1 and AD, there is only one pKa value at ~8 observed in the pH dependence profile. There is a small difference of 0.2 pH units between the estimated pKa values of the kcat and kcat/KmAD curves (Fig. 5), but the values are not significantly different considering the experimental variance. Tyr9 of GST A1-1 has been assigned a pKa value of 8.1 in the absence of GSH and a pKa value of 9.2 in the presence of GSH (41). These figures correlate well with the pKa values observed in this work and lead to the hypothesis that the side chain of Tyr9 is the second of the ionizable groups of GST A1-1 catalyzing the isomerization reaction. In the presence of GSH, serving as a base, Tyr9 promotes catalysis in its protonated form. However, in the absence of GSH, Tyr9 seems to adopt the role of the base in the catalytic mechanism. A structure of GST A1-1 with AD bound is not available, but the sulfur of GSH and the phenolic oxygen of Tyr9 are adjacent in the known GST A1-1 structures (14). Thus, both of the proposed functional groups may interact with the reactive region of the AD molecule, allowing for alternative roles of Tyr9 in catalysis.
A comparison of GST A1-1 and the active-site tyrosine mutant Y9F first demonstrates that the kcat value for the isomerization of AD is decreased 48 times in Y9F (Table II). The KmGSH value is lowered 11 times, whereas the KmAD value is lowered only two times in Y9F. The kcat/KmGSH and kcat/KmAD values for wild-type GST A1-1 are 4 and 21 times higher, respectively, than the corresponding parameters of Y9F.
Nonproductive binding of a substrate is expected to decrease both kcat and Km by the same factor, leaving kcat/Km unchanged (42). Thus, the marked effects of the Y9F mutation on kcat and KmGSH suggest that GSH to a large extent is bound in a catalytically nonproductive mode in the mutant. In wild-type GST A1-1 the proposed hydrogen bond between the sulfur of GSH and the phenolic hydroxyl group of Tyr9 (14) could help orienting the orbitals of the thiolate for optimal catalytic efficiency. The loss of this steering effect in Y9F may account for a major portion of the decreased activity. However, it is clear that also other factors are involved.
The enzyme-bound GSH thiol group has a pKa value that is somewhat higher for Y9F (7.2) than the corresponding value (6.7) for wild-type GST A1-1. This is in agreement with the finding that the hydroxyl group of the active-site tyrosine in a rat Mu class GST contributes to the stabilization of the thiolate in the enzyme-GSH complex (43). However, the shift of the pKa value for GSH does not fully account for the altered pH profiles of the kinetic parameters kcat and kcat/KmAD with pKa values for Y9F 8.2 and 8.4, respectively (Fig. 5). This discrepancy suggests a pH-dependent change in rate-determining step or that at least one additional functional group is partaking in catalysis.
The kcat/KmGSH and the kcat/KmAD values of GST A2-2 are both lower than the corresponding values of GST A1-1 and Y9F. The kcat/KmGSH value is 36 times and the kcat/KmAD value is 480 times lower than for GST A1-1. By this measure GST A2-2 differs from Y9F about as much as Y9F is differing from GST A1-1. This finding contrasts with the notion that Tyr9, present in all the naturally occurring Alpha class isoenzymes, should play an important role in the isomerization of AD, in analogy with its significance in conjugation reactions. For example, GST A2-2 has a reasonably high specific activity with CDNB, while Y9F does not (Table II). Again the results indicate that there are amino acid residues in addition to Tyr9 that are important for the isomerization reaction.
GST A2-2 differs from GST A1-1 by only 11 amino acids out of a total of 221 in a protein subunit. Four of these are situated in the active site and all of them in the H-site. The four mutations in GST A2-2 required to mimic the active site of GST A1-1 are S10F, I12A, F111V, and S216A. The two serine residues in GST A2-2, Ser10 and Ser216, provide a less hydrophobic environment and may afford hydrogen bond interactions between substrate and enzyme. Both GST A1-1 and GST A2-2 have a phenylalanine in the active site but in different positions, Phe10 and Phe111, respectively. In position 111 the residue may be too bulky, whereas in position 10 phenylalanine may actually contribute to productive binding of AD as seen for the Pseudomonas 3-ketosteroid isomerase (44). Furthermore, an isoleucine residue (Ile12) next to the active site tyrosine in GST A2-2 may, for example, sterically hinder the large AD molecule, whereas the same position in GST A1-1 is occupied by the much less bulky alanine. The other seven differences between GST A1-1 and A2-2 involve residues that probably do not interact with the bound AD, even though they might still have an indirect influence on catalysis.
The reaction rate of GST A4-4 with AD is 3 orders of magnitude lower than that of GST A1-1. The structural differences between GST A1-1 and GST A4-4 are extensive, and their amino acid sequence similarity is only 53% overall. Thus, it is noteworthy that there is a measurable activity with AD despite the significant differences in primary structures of GST A4-4 and the other isoenzymes investigated in this work. The KmGSH and KmAD values of GST A4-4 are similar to the corresponding values of GST A1-1, and the major difference in catalytic efficiency between GST A4-4 and GST A1-1 is a reflection of the kcat values that differ by 3 orders of magnitude.
The bacterial 3-ketosteroid isomerase identified in
Pseudomonas (1) is one of the most efficient enzymes known.
Its catalytic mechanism has been studied in great detail. A key residue
of the active site is Asp38, the carboxylate that shuttles
a proton from C4 to C6 of the substrate AD in the isomerization
reaction (45). A tyrosine residue, Tyr14, assisted by
Asp99 promotes the isomerization by hydrogen bonding and
polarization of the 3-keto group of AD (46, 47). Little is known about the catalytic mechanism of the bifunctional mammalian 3-HSD/Iso, but
it is noteworthy that mutational studies have shown two neighboring tyrosine residues, Tyr253 and Tyr254, to be
important for the isomerase activity (48).
The present study demonstrates the importance of two functional groups for the isomerization of AD catalyzed by GST A1-1. The most prominent role is played by the sulfhydryl group of GSH bound as a cofactor in the active site. It acts in its basic thiolate form and presumably relays a proton from C4 to C6 in AD, thus playing a similar role as Asp38 in the bacterial isomerase. The second functional group is the phenolic hydroxyl of Tyr9, which is active in its protonated form. This group may promote catalysis not only by stabilizing the GSH thiolate but also by polarizing the 3-keto group of AD. In the absence of GSH Tyr9 takes over as a base instead of the thiolate of GSH, as indicated by the pH activity profile (Fig. 5) and the lack of activity of Y9F in the absence of GSH.
The primary goal of the present study was to clarify the role of GSH in
the double-bond isomerization reaction, which is quite distinct from
the previously investigated GST-catalyzed conjugation reactions leading
to detoxication and excretion of reactive electrophiles. However,
several lines of evidence suggest that the steroid isomerization effected by GST A1-1 may be physiologically important in mammalian tissues. Pregnenolone is an obligatory intermediate in the biosynthesis of steroid hormones, and in the available alternative metabolic pathways its 3-hydroxyl group will eventually be oxidized to a
ketone, and its
5 double bond will be converted to the
resonance-stabilized
4 isomer. For example, a reaction
sequence leading to testosterone involves the 3
-oxidation of
dehydroepiandrosterone to
5-androstenedione followed by
the isomerization of the product into
4-androstenedione
(2). In mammalian tissues the oxidation is catalyzed by several
pyridine-nucleotide-dependent 3
-hydroxysteroid dehydrogenases. Of the six homologous dehydrogenases identified so far,
four NAD+-dependent dehydrogenases are believed
to be involved in the biosynthesis of steroid hormones (49). One of
them is the principal form in gonads and adrenal glands. Other
isoenzymes are expressed in organs such as liver and kidney. Although
progress has been made in the study of the differential expression of
the multiple forms in adult and fetal tissues, many issues concerning
the catalytic properties and the physiological roles of the distinct
isoenzymes remain unresolved. In Pseudomonas the
3
-hydroxysteroid dehydrogenase is accompanied by a highly efficient
5-3-ketosteroid isomerase (1), and the two enzymes can
be separated by chromatography. A similar efficient isomerase has not
been identified in mammalian tissues, and at least some of the
mammalian dehydrogenases have been shown to have intrinsic steroid
isomerase activity (4, 49) and are therefore referred to as
bifunctional 3
-hydroxysteroid
dehydrogenase/
5
4 isomerases. The
dehydrogenase reaction can be studied separately from the isomerization
and vice versa, and it has been suggested that the two distinct
catalytic activities reside in distinct active sites (48, 51). However,
unless there is efficient channeling of the product of the oxidation to
the site of the isomerization reaction, there may be substantial
leakage of the
5-intermediate to the surrounding medium.
The higher Km values for the dehydrogenase expressed
in liver and kidney as compared with that found in gonads and adrenal
glands (52) suggest that the need for a supplementary steroid isomerase
may be particularly high in the former organs.
The present study demonstrates that human GST A1-1 is highly efficient
in catalyzing the isomerization of AD. This enzyme is one of the most
abundant proteins in liver, kidney, and testis, in which it may exceed
2% of the total soluble protein (53, 54). The catalytic efficiency of
GST A1-1, 50·104 s1
M
1, determined in the present
investigation, is obtained at saturating GSH levels (millimolar
concentrations), which are achieved in most tissues under normal
physiological conditions. For comparison, kinetic parameters are
available for 3
-HSD/Iso from human placenta (48) with a calculated
specificity constant
(kcat/Km) of
1.6·104 s
1
M
1 with AD as the substrate. This
value is 30-fold lower than that of GST A1-1. In view of the
exceedingly high intracellular concentration of GST A1-1, its isomerase
activity with AD may exceed by several orders of magnitude that of the
3
-hydroxysteroid dehydrogenase, particularly in tissues such as
liver and kidney. Recent
studies7 have shown that GST
A1-1 is almost as active with
5-pregnen-3,20-dione, an
intermediate on the pathway to progesterone.
In conclusion, the GST-catalyzed isomerization of AD provides a
complement to the enzyme reactions previously associated with 3-hydroxysteroid dehydrogenases in mammalian tissues. The steroid isomerization is one of the most efficient of the known GST-catalyzed reactions involving an endogenous substrate. Other examples include 4-hydroxynonenal (32, 55) and ortho-quinones derived from catecholamines (50, 56), compounds that originate by oxidative processes. The common denominator for these reactions is that only one
of the multiple homologous forms of GST has the characteristic high
activity with the endogenous substrate, suggesting that the activity
has evolved specifically for the catalyzed reaction.
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ACKNOWLEDGEMENTS |
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We are grateful to Ann Gustafsson, Dr. Ina Hubatsch, Dr. Per Jemth, Dr. Gun Stenberg, and Dr. Mikael Widersten for providing expression clones, enzymes, GSHex, and scientific advice.
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FOOTNOTES |
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* This work was supported by the Swedish Natural Science Research Council.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.: 46-18-4714539;
Fax: 46-18-558431; E-mail: Bengt.Mannervik@biokem.uu.se.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M009146200
2 The nomenclature system for GSTs is presented in Ref. 7.
3 A. Gustafsson, and B. Mannervik, unpublished data.
4 K. Svensson, M. Widersten, and B. Mannervik, unpublished data.
5 I. Hubatsch, personal communication.
6 Reaction rate data derived from Fig. 5A.
7 A.-S. Johansson and B. Mannervik, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
AD, 5-androstene-3,17-dione;
GST, glutathione transferase;
GSH, glutathione;
CDNB, 1-chloro-2,4-dinitrobenzene;
GSMe, S-methylglutathione;
o-CF3CDNB,
2-chloro-3,5-dinitro-1,1,1-(trifluoromethyl)benzene;
GSHex, S-hexylglutathione;
Y9F, GST A1-1 mutant Y9F;
3
-HSD/Iso, 3
-hydroxysteroid
dehydrogenase/
5
4Isomerase;
H-site, hydrophobic site;
G-site, GSH binding site.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Talalay, P., and Wang, V. S. (1955) Biochim. Biophys. Acta 18, 300-301[CrossRef][Medline] [Order article via Infotrieve] |
2. | Samuels, L. T., Helmreich, M. L., Lasater, M. B., and Reich, H. (1951) Science 113, 490-491[Medline] [Order article via Infotrieve] |
3. |
Ford, H. C.,
and Engel, L. L.
(1974)
J. Biol. Chem.
249,
1363-1368 |
4. | Thomas, J. L., Myers, R. P., and Strickler, R. C. (1989) J. Steroid Biochem. 33, 209-217[CrossRef][Medline] [Order article via Infotrieve] |
5. | Benson, A. M., and Talalay, P. (1976) Biochem. Biophys. Res. Commun. 69, 1073-1079[Medline] [Order article via Infotrieve] |
6. | Benson, A. M., Talalay, P., Keen, J. H., and Jakoby, W. B. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 158-162[Abstract] |
7. | Mannervik, B., Awasthi, Y. C., Board, P. G., Hayes, J. D., Di Ilio, C., Ketterer, B., Listowsky, I., Morgenstern, R., Muramatsu, M., Pearson, W. R., Pickett, C. B., Sato, K., Widersten, M., and Wolf, C. R. (1992) Biochem. J. 282, 305-306[Medline] [Order article via Infotrieve] |
8. | Mannervik, B., Ålin, P., Guthenberg, C., Jensson, H., Tahir, M. K., Warholm, M., and Jörnvall, H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7202-7206[Abstract] |
9. | Meyer, D. J., Coles, B., Pemble, S. E., Gilmore, K. S., Fraser, G. M., and Ketterer, B. (1991) Biochem. J. 274, 409-414[Medline] [Order article via Infotrieve] |
10. | Meyer, D. J., and Thomas, M. (1995) Biochem. J. 311, 739-742[Medline] [Order article via Infotrieve] |
11. | Pemble, S. E., Wardle, A. F., and Taylor, J. B. (1996) Biochem. J. 319, 749-754[Medline] [Order article via Infotrieve] |
12. | Board, P. G., Baker, R. T., Chelvanayagam, G., and Jermiin, L. S. (1997) Biochem. J. 328, 929-935[Medline] [Order article via Infotrieve] |
13. |
Board, P. G.,
Coggan, M.,
Chelvanayagam, G.,
Easteal, S.,
Jermiin, L. S.,
Schulte, G. K.,
Danley, D. E.,
Hoth, L. R.,
Griffor, M. C.,
Kamath, A. V.,
Rosner, M. H.,
Chrunyk, B. A.,
Perregaux, D. E.,
Gabel, C. A.,
Geoghegan, K. F.,
and Pandit, J.
(2000)
J. Biol. Chem.
275,
24798-24806 |
14. | Sinning, I., Kleywegt, G. J., Cowan, S. W., Reinemer, P., Dirr, H. W., Huber, R., Gilliland, G. L., Armstrong, R. N., Ji, X., Board, P. G., Olin, B., Mannervik, B., and Jones, T. A. (1993) J. Mol. Biol. 232, 192-212[CrossRef][Medline] [Order article via Infotrieve] |
15. | Stenberg, G., Board, P. G., and Mannervik, B. (1991) FEBS Lett. 293, 153-155[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Ji, X.,
von Rosenvinge, E. C.,
Johnson, W. W.,
Armstrong, R. N.,
and Gilliland, G. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
8208-8213 |
17. | McTigue, M. A., Williams, D. R., and Tainer, J. A. (1995) J. Mol. Biol. 246, 21-27[CrossRef][Medline] [Order article via Infotrieve] |
18. | Mannervik, B., and Danielson, U. H. (1988) CRC Crit. Rev. Biochem. 23, 283-337[Medline] [Order article via Infotrieve] |
19. | Hayes, J. D., and Pulford, D. J. (1995) Crit. Rev. Biochem. Mol. Biol. 30, 445-600[Abstract] |
20. | Armstrong, R. N. (1997) Chem. Res. Toxicol. 10, 2-18[CrossRef][Medline] [Order article via Infotrieve] |
21. | Tsuchida, S., and Sato, K. (1992) Crit. Rev. Biochem. Mol. 27, 337-384 |
22. | Tew, K. D. (1994) Cancer Res. 54, 4313-4320[Abstract] |
23. |
Dragani, B.,
Stenberg, G.,
Melino, S.,
Petruzzelli, R.,
Mannervik, B.,
and Aceto, A.
(1997)
J. Biol. Chem.
272,
25518-25523 |
24. | Dirr, H., Reinemer, P., and Huber, R. (1994) Eur. J. Biochem. 220, 645-661[Abstract] |
25. | Wilce, M. C. J., and Parker, M. W. (1994) Biochim. Biophys. Acta 1205, 1-18[Medline] [Order article via Infotrieve] |
26. | Hansson, L. O., Widersten, M., and Mannervik, B. (1997) Biochemistry 36, 11252-11260[CrossRef][Medline] [Order article via Infotrieve] |
27. | Hansson, L. O., Bolton-Grob, R., Massoud, T., and Mannervik, B. (1999) J. Mol. Biol. 287, 265-276[CrossRef][Medline] [Order article via Infotrieve] |
28. | Stenberg, G., Björnestedt, R., and Mannervik, B. (1992) Protein Expression Purif. 3, 80-84[Medline] [Order article via Infotrieve] |
29. | Rhoads, D. M., Zarlengo, R. P., and Tu, C.-P. D. (1987) Biochem. Biophys. Res. Commun. 145, 474-480[Medline] [Order article via Infotrieve] |
30. | Widersten, M., and Mannervik, B. (1995) J. Mol. Biol. 250, 115-122[CrossRef][Medline] [Order article via Infotrieve] |
31. | Gustafsson, A., and Mannervik, B. (1999) J. Mol. Biol. 288, 787-800[CrossRef][Medline] [Order article via Infotrieve] |
32. | Hubatsch, I., Ridderström, M., and Mannervik, B. (1998) Biochem. J. 330, 175-179[Medline] [Order article via Infotrieve] |
33. | Widersten, M., Björnestedt, R., and Mannervik, B. (1994) Biochemistry 33, 11717-11723[Medline] [Order article via Infotrieve] |
34. | Bardsley, W. G., Bukhari, N. A. J., Ferguson, M. W. J., Cachaza, J. A., and Burguillo, F. J. (1995) Comput. Chem. 19, 75-84[CrossRef] |
35. | Widersten, M., Björnestedt, R., and Mannervik, B. (1996) Biochemistry 35, 7731-7742[CrossRef][Medline] [Order article via Infotrieve] |
36. | Cleland, W. W. (1963) Biochim. Biophys. Acta 67, 104-137[CrossRef] |
37. | Gustafsson, A., Etahadieh, M., Jemth, P., and Mannervik, B. (1999) Biochemistry 38, 16268-16275[CrossRef][Medline] [Order article via Infotrieve] |
38. | Barycki, J. J., and Colman, R. F. (1997) Arch. Biochem. Biophys. 345, 16-31[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Wang, J.,
Bauman, S.,
and Colman, R. F.
(2000)
J. Biol. Chem.
275,
5493-5503 |
40. | Fersht, A. (1999) Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding , p. 178, W. H. Freeman and Co., New York |
41. | Björnestedt, R., Stenberg, G., Widersten, M., Board, P. G., Sinning, I., Jones, A., and Mannervik, B. (1995) J. Mol. Biol. 247, 765-773[CrossRef][Medline] [Order article via Infotrieve] |
42. | Fersht, A. (1999) Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding , pp. 114-117, W. H. Freeman and Co., New York |
43. |
Liu, S.,
Zhang, P.,
Ji, X.,
Johnson, W. W.,
Gilliland, G. L.,
and Armstrong, R. N.
(1992)
J. Biol. Chem.
267,
4296-4299 |
44. | Brothers, P. N., Blotny, G., Qi, L., and Pollack, R. M. (1995) Biochemistry 34, 15453-15458[Medline] [Order article via Infotrieve] |
45. | Kuliopulos, A., Westbrook, E. M., Talalay, P., and Mildvan, A. S. (1987) Biochemistry 26, 3927-3937[Medline] [Order article via Infotrieve] |
46. | Massiah, M. A., Abeygunawardana, C., Gittis, A. G., and Mildvan, A. S. (1998) Biochemistry 37, 14701-14712[CrossRef][Medline] [Order article via Infotrieve] |
47. | Thornburg, L. D., Hénot, F., Bash, D. P., Hawkinson, D. C., Bartel, S. D., and Pollack, R. M. (1998) Biochemistry 37, 10499-10506[CrossRef][Medline] [Order article via Infotrieve] |
48. | Thomas, J. L., Evans, B. W., Blanco, G., Mercer, R. W., Mason, J. I., Adler, S., Nash, W. E., Isenberg, K. E., and Strickler, R. C. (1998) J. Steroid Biochem. Mol. Biol. 66, 327-334[CrossRef][Medline] [Order article via Infotrieve] |
49. | Payne, A. H., Abbaszade, I. G., Clarke, T. R., Bain, P. A., and Park, C.-H. (1997) Steroids 62, 169-175[CrossRef][Medline] [Order article via Infotrieve] |
50. | Baez, S., Segura-Aguilar, J., Widersten, M., Johansson, A.-S., and Mannervik, B. (1997) Biochem. J. 324, 25-28[Medline] [Order article via Infotrieve] |
51. | Luu-The, V., Takahashi, M., de Launoit, Y., Dumont, M., Lachance, Y., and Labrie, F. (1991) Biochemistry 30, 8861-8865[Medline] [Order article via Infotrieve] |
52. | Clarke, T. R., Bain, P. A., Sha, L., and Payne, A. H. (1993) Endocrinology 132, 1971-1976[Abstract] |
53. | van Ommen, B., Boogards, J. J. P., Peters, W. H. M., Blaauboer, B., and van Bladeren, P. J. (1990) Biochem. J. 269, 609-613[Medline] [Order article via Infotrieve] |
54. | Rowe, J. D., Nieves, E., and Listowsky, I. (1997) Biochem. J. 325, 481-486[Medline] [Order article via Infotrieve] |
55. | Stenberg, G., Ridderström, M., Engström, Å., Pemble, S. E., and Mannervik, B. (1992) Biochem. J. 284, 313-319[Medline] [Order article via Infotrieve] |
56. |
Segura-Aguilar, J.,
Baez, S.,
Widersten, M.,
Welch, C. J.,
and Mannervik, B.
(1997)
J. Biol. Chem.
272,
5727-5731 |