(Received for publication, May 19, 1995; and in revised form, July 7, 1995)
From the
Monobromobimane (mBBr), besides being a substrate in the
presence of glutathione, inactivates rat liver glutathione S-transferase 3-3 at pH 7.5 and 25 °C as assayed
using 1-chloro-2,4-dinitrobenzene (CDNB). The rate of inactivation is
enhanced about 5-fold by S-methylglutathione. Substrate
analogs bromosulfophthalein and 2,4-dinitrophenol decrease the rate of
inactivation at least 20-fold. Upon incubation for 60 min with 0.25
mM mBBr and S-methylglutathione, the enzyme loses 91%
of its activity toward CDNB and incorporates 2.14 mol of reagent/mol of
subunit, whereas incubation under the same conditions but with added
protectant 2,4-dinitrophenol yields an enzyme that is catalytically
active and contains only 0.89 mol of reagent/mol of subunit.
mBBr-modified enzyme is fluorescent, and fluorescence energy transfer
occurs between intrinsic tryptophan and covalently bound bimane in
modified enzyme. Both Tyr and Cys
are
modified, but Tyr
is the initial reaction target and its
modification correlates with loss of activity toward CDNB. The fact
that the activity toward mBBr is retained by the enzyme after
modification suggests that rat isozyme 3-3 has two binding sites for mBBr.
Glutathione S-transferases constitute a family of
enzymes that catalyze the nucleophilic attack by the thiol of
glutathione on a variety of structurally diverse endogenic and
xenobiotic
substrates(1, 2, 3, 4, 5, 6) .
Glutathione S-transferases are classified into five classes,
one microsomal and four cytosolic (, µ,
, and
)(2, 6, 7) . Each class consists of two
or more different subunits, which combine to form homodimers or
heterodimers. Crystallographic studies of
, µ, and
isozymes indicate that each protein subunit has two distinct
domains(8, 9, 10, 11, 12, 13) .
The glutathione binding site is located almost entirely within the N-terminal domain with only salt bridges at each end of the
tripeptide linking to domain II of the adjacent subunit, while the
xenobiotic binding site is a highly hydrophobic pocket composed of
residues in both the N- and C-terminal
domains(11, 14) . These crystallographic studies
coupled with site-directed mutagenesis and kinetic studies of the
enzyme have provided some insights into the structural importance of
amino acid residues or regions in catalysis and binding of glutathione
and certain xenobiotic substrates(6, 9, 11) .
However, little is known about the binding of xenobiotic substrates to
the enzyme and what determines the distinct substrate specificities of
the various isozymes of glutathione S-transferase.
Among
the residues contributing to the binding of substrates and catalysis of
the enzyme is a tyrosine residue in the N-terminal region of the
enzyme: i.e. Tyr in 1-1 and Tyr
in 3-3 and 4-4 isozymes. The tyrosine hydroxyl group
is within hydrogen bonding distance (3.2-3.4 Å) of the
sulfur of glutathione and is thought to be responsible for decreasing
the pK
of the sulfhydryl group of
glutathione(9, 15) . Affinity labeling studies using S-(4-bromo-2,3-dioxobutyl)glutathione and
4-(fluorosulfonyl)benzoic acid demonstrated that Tyr
in
both isozyme 3-3 and 4-4 is located in the xenobiotic
binding site and contributes to binding of these hydrophobic substrates
in addition to its function as a general acid in reactions with certain
substrates (i.e. those involving epoxide ring opening and
Michael addition reactions)(11, 16, 17) .
More recently, Tyr
has been identified as a target of
other affinity labels(18, 19) . Reaction of isozyme
3-3 with S-(4-bromo-2,3-dioxobutyl)glutathione
demonstrated that Cys
in this isozyme is also modified but
without loss of activity when the reaction is carried out in the
presence of S-hexylglutathione(16) ; this result is
consistent with the crystal structure of the enzyme, which shows that
Cys
is located on the loop connecting the two domains and
is exposed to solvent(9, 10, 11) .
Monobromobimane (mBBr) ()is a nonfluorescent hydrophobic
compound that reacts with nucleophiles to give fluorescent
derivatives(20) . mBBr was reported to be a substrate of an
unfractionated preparation of glutathione S-transferases,
although activities of individual isozymes were not
distinguished(21) .
In this paper, we report that mBBr, besides being a substrate of rat liver glutathione S-transferase 3-3, also acts as an affinity label of this isozyme in the presence of a glutathione analog, S-methylglutathione. A series of experiments are described that are designed to determine the amino acid and functional targets of mBBr within glutathione S-transferase 3-3. We report that covalent modification with mBBr causes substantial loss of enzymatic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) with little or no effect toward another substrate, suggesting the existence of more than one binding site for xenobiotic substrates in the 3-3 isozyme. A preliminary version of this work was presented at the Eighth Symposium of the Protein Society(22) .
mB-Cys was prepared by reacting mBBr with 20-fold excess cysteine at pH 8. Briefly, a 50 mM mBBr solution in DMF (2 µl) was added to a 1 mM cysteine solution in 50 mM ammonium bicarbonate at pH 8 (2 ml). The mixture was stirred at room temperature for 5 h, and the product was purified by HPLC using the same system as for mB-Tyr, with mB-Cys eluting at 14% acetonitrile. The UV absorption spectrum for mB-Cys shows a wavelength maximum at 390 nm. The fluorescence spectra exhibit an excitation maximum at 395 nm and an emission maximum at 480 nm, similar to that of mB-SG in terms of the position of the peaks and in fluorescence intensity at comparable concentrations. The PTH derivative of mB-Cys is a distinct peak appearing between PTH-Tyr and PTH-Pro on an Applied Biosystems gas-phase protein (peptide) sequencer; the amount of mB-Cys was estimated using Met as a standard.
Other
substrates utilized to assay for enzymatic activity include
bromosulfophthalein and mBBr. For bromosulfophthalein, the enzymatic
activity was measured at 330 nm ( = 4.5
mM
cm
) by monitoring the
conjugate formation between bromosulfophthalein (0.2 mM) and
glutathione (5.0 mM) in 0.1 M potassium phosphate
buffer, pH 7.5, at 25 °C according to the method of Habig et
al.(31) . For mBBr, the enzymatic activity was measured
using a Perkin-Elmer MPF-3 fluorescence spectrophotometer (excitation
at 395 nm and emission at 480 nm) by monitoring the formation of the
conjugate of mBBr (30 µM) and glutathione (600
µM) in 0.1 M potassium phosphate buffer, pH 6.5,
at 25 °C according to the method of Hulbert and
Yakubu(21) . A lower glutathione concentration was chosen
because of the relatively large spontaneous nonenzymatic rate of
reaction between glutathione and mBBr. For each experiment, a known
amount of mB-SG was prepared in the presence of glutathione S-transferase (0.5 µg/ml) from mBBr (5 µM)
and glutathione (600 µM) and used as a fluorescence
standard to calibrate and calculate the initial rate of product
formation.
To determine the apparent K value of
glutathione, a range of glutathione concentrations (9-600
µM) was investigated at a constant mBBr concentration (100
µM). Similarly, the apparent K
for
mBBr was determined at a range of concentrations of mBBr (0.4-100
µM) and a constant concentration of glutathione (600
µM). Data were analyzed by fitting directly to the
Michaelis-Menten equation of v = V
C/(K
+ C) using a nonlinear curve fitting program MINSQ from
MicroMath Scientific Software.
In the preparation of modified and control enzyme, excess unreacted reagent was removed from the reaction mixture by the gel filtration procedure of Penefsky(32) . Aliquots (0.5 ml) of the reaction mixture at the end of reaction were applied to two successive 5-ml columns of Sephadex G-50 equilibrated with 0.1 M potassium phosphate buffer, pH 7.5; it was found later that one column was sufficient to remove the unreacted reagent. The protein concentration in the filtrate was determined by the Bio-Rad protein assay, which is based on the dye-binding method of Bradford(33) , using a Bio-Rad 2550 radioimmunoassay reader (600-nm filter). Purified glutathione S-transferase 3-3 was used to establish the standard protein concentration curve for these determinations.
After dialysis, the solution of modified enzyme was lyophilized. The lyophilized enzyme was solubilized in 8 M urea in 50 mM ammonium bicarbonate (250 µl) by incubation at 37 °C for 2 h, after which 750 µl of 50 mM ammonium bicarbonate was added to give a final urea concentration of 2 M. The modified glutathione S-transferase was digested at 37 °C with two additions of N-tosyl-L-phenylalanine chloromethylketone-treated trypsin (2.5% w/w) at 1-h intervals. After trypsin digestion, the digest was lyophilized and stored at -20 °C.
When further purification of peptides was needed, samples were separated using a second solvent system with 20 mM ammonium acetate in water, pH 6.0, as solvent A and 20 mM ammonium acetate in 90% acetonitrile, pH 6.0, as solvent B. Elution was started with isocratic 20% solvent B for 5 min followed by a linear gradient to 100% solvent B for a total of 149 min at a flow rate of 1 ml/min (chromatography system 2).
Figure 1:
Kinetics of the formation of the
glutathione conjugate of mBBr as catalyzed by glutathione S-transferase 3-3. Rat liver glutathione S-transferase 3-3 (0.3 µg/ml) was incubated in the
presence of 600 µM glutathione and various concentrations
of mBBr at pH 6.5 and 25 °C, and the product formation was
monitored by fluorescence as described under ``Experimental
Procedures.'' The data, fitted to the Michaelis-Menten equation,
gave K = 0.54 µM and V
= 3.4 µmol/min/mg. Inset, Lineweaver-Burk plot of 1/vversus 1/[mBBr].
Figure 2:
Inactivation of glutathione S-transferase 3-3 by mBBr. Rat liver glutathione S-transferase 3-3 (0.3 mg/ml) was incubated with
() or without (
) 2 mM mBBr at pH 7.5 and 25 °C.
Residual activity, E
/E
, was measured as
described under ``Experimental Procedures.'' Inset, the concentration dependence of inactivation of glutathione S-transferase 3-3 by mBBr appears to be linear with a
second order rate constant of 0.026 min
mM
.
Figure 3:
Inactivation of glutathione S-transferase 3-3 by mBBr in the presence of 5 mMS-methylglutathione. Rat liver glutathione S-transferase 3-3 (0.3 mg/ml) was incubated with various
concentrations of mBBr in the presence of 5 mMS-methylglutathione at pH 7.5 and 25 °C. Residual
activity E/E
was
measured as described under ``Experimental Procedures.''
Concentrations of mBBr shown are 0.15 mM (
), 0.25 mM (
), and 1 mM (
). Inset, plot of the
observed rate constant of inactivation of glutathione S-transferase 3-3 by mBBr in the presence of 5
mMS-methylglutathione versus the
concentration of mBBr. The data, fitted to equation k
= (k
)/(K
+
[mBBr]), gave k
= 0.56
min
and K
=
1.73 mM, with a k
/K
= 0.32 min
mM
.
Figure 4:
HPLC separation of proteolytic digests of
proteins resulting from the 60-min modification of glutathione S-transferase 3-3 by mBBr. The enzyme was modified by
0.25 mM mBBr in the presence of 5 mMS-methylglutathione for 60 min, digested, and separated
as described under ``Experimental Procedures.'' A (A) and B (A
) show profiles of digest of the modified enzyme prepared in
the absence of protectant 2,4-dinitrophenol. C, A
profile of digest of the modified enzyme
prepared in the presence of protectant
2,4-dinitrophenol.
Fig. 4C shows the HPLC pattern of a tryptic digest, as monitored at 390 nm, of catalytically active, modified glutathione S-transferase prepared by incubation of the rat liver enzyme for 60 min with 0.25 mM mBBr in the presence of 5 mMS-methylglutathione and 10 mM 2,4-dinitrophenol. The digest of this active enzyme (Fig. 4C) contains no significant amount of peptides corresponding to peaks I-IV of the inactive enzyme digest (Fig. 4B); the measured reagent incorporation of 0.89 mol/mol of subunit was distributed in small amounts at different nonspecific sites as shown using the more sensitive measurement of fluorescence associated with each peptide (data not shown). These results indicate that peaks I, Ia, II, III, and IV contain modified peptides, whose appearance correlates with inactivation.
The development of these modified peptide peaks was monitored as a function of time of reaction and corresponding degree of inactivation. The enzyme was 35% inactivated after incubation for 8 min with 0.25 mM mBBr in the presence of 5 mMS-methylglutathione; in this sample, peak II was the most abundant modified peptide present with only traces of I and III. Upon increasing the incubation time to 15 min (yielding 55% inactivated enzyme) and to 60 min (yielding 91% inactivated enzyme), peak II decreased in intensity while peaks I, III, and IV clearly increased, indicating that peak II is the precursor of one or more of four later-appearing peaks (I, Ia, III, and IV).
Peptide I and Ia
represent different proteolytic products of the same peptide, where
both Cys and Tyr
are modified by mBBr. In
peptide II, only Tyr
is modified by mBBr, while
Cys
is not modified by mBBr and is available for
subsequent N-ethylmaleimide modification. Only Cys
is modified by mBBr in peptide III. In peptide IV, at both cycles
7 and 8, the amino acids are identified to be mB-Cys while the rest of
the amino acids are identical to those in peptide I, II, and III,
suggesting the existence of a new form of rat liver glutathione S-transferase 3-3 with a Tyr to Cys mutation at position
115. The relative amount of this mutated form is batch-dependent (data
not shown). Such a mutation at an equivalent position of the rat
glutathione S-transferase µ class subunit Yb
was reported previously by others (37, 38) and
resulted in an enzyme that was shown to be difficult to separate from
the other µ class isozymes(38) .
Figure 5: Fluorescence emission spectra of modified glutathione S-transferase 3-3 under nondenaturing conditions at pH 7.5 and 25 °C (--) and denaturing conditions in 4 M guanidine HCl, pH 7.5, and 25 °C(- - -). Modified glutathione S-transferase was prepared by reacting the enzyme (0.3 mg/ml) with 0.25 mM mBBr in the presence of 5 mMS-methylglutathione at pH 7.5 and 25 °C, and the samples were excited at 280 nm as described under ``Experimental Procedures.''
The apparent K and V
values of modified and control enzymes for
mBBr and glutathione were determined in order to further evaluate the
effect of modification. As shown in Table 5, the modification
affects both the apparent K
and V
, with the effect on K
being much greater than that on V
, and the
effect on mBBr being greater than that on glutathione. For mBBr, the K
was increased by 50-fold, and V
was also increased by about 3-fold. Overall, the catalytic
efficiency of the modified enzyme decreased by about 15-fold as judged
by the apparent k
/K
. For
glutathione, the K
was increased by about 8-fold
and V
by about 2.3-fold, resulting in an overall
decrease in the catalytic efficiency by 3.6-fold.
The results of this paper demonstrate that mBBr reacts
predominantly with 2 amino acid residues of glutathione S-transferase 3-3: Tyr and
Cys
. However, modification occurs initially at
Tyr
, paralleling the loss of enzymatic activity toward
CDNB. Cys
modification appears to be slow and secondary,
and it is not clear whether modification of only this residue is
sufficient to cause inactivation. A schematic representation of the
sequence of reaction of mBBr with glutathione S-transferase is
shown in .
From the time-dependent development of modified peptides, we
conclude that the rate of reaction of mBBr at Tyr of the
native enzyme, k
, is greater than the reaction
rate at Cys
of the native enzyme, k
.
The accumulation of peptide II and the slow appearance of peptide I
(and Ia) at the early period of reaction indicate that k
is greater than k
. The slow appearance and
the existence of significant amount of peptide III peak at 60 min
indicates that modification of Cys
slows down further
modification at Tyr
, i.e.k
> k
. Therefore, the relative order of the
rate constants for modification is k
> k
> k
and k
> k
. Cys
is not appreciably
modified by mBBr in our studies, although it does react with S-(4-bromo-2,3-dioxobutyl)glutathione(16) . This
difference could be due to the more hydrophobic character of mBBr,
which makes it more likely to react within the interior of proteins.
From the crystal structure represented in Fig. 6, Tyr is positioned in a conformation to react with mBBr docked at the
active site, which would explain why this residue is modified first and
the rate of reaction is greater. Fig. 6also suggests that
Cys
is located in the vicinity of the active site; but
the sulfur atom of this cysteinyl residue is pointed away from the
substrate binding cavity, making it inaccessible to the active
site-bound reagent. As suggested in the affinity labeling studies of
glutathione S-transferase 3-3 using
2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone(18) ,
a conformation different from the one shown in Fig. 6may exist
in solution, in which the cysteinyl side chain is more exposed to the
active site-bound reagent.
Figure 6:
A model showing the relative locations of
side chains of Tyr and Cys
in one of the
docked structures of glutathione S-transferase 3-3 and
mBBr. Glutathione S-transferase 3-3 is illustrated as redribbons. Atoms of protein side chains are shown
in white, those for S-methylglutathione in yellow, and those for mBBr in blue. The whitearrow indicates the attack of the phenolic oxygen
nucleophile on the bromomethyl carbon of mBBr docked in the xenobiotic
binding site of glutathione S-transferase
3-3.
Alternatively, the sulfhydryl group of
Cys may react with mBBr from the outside of the protein
molecule. In the structure shown in Fig. 6, this sulfhydryl
group is imbedded in a hydrophobic environment
7-8 Å
from the surface, surrounded by the hydrophobic side chains of
Trp
, Phe
, Phe
,
Phe
, and Leu
. Close examination of the
structure reveals that a bimane molecule can be placed in a position to
react with the sulfhydryl group of Cys
, requiring minimal
perturbation of conformations of residues in this region. This is
consistent with the results of energy minimization of a model (not
shown) of glutathione S-transferase 3-3 with covalently
bound bimanes at both Tyr
and Cys
, which
leads only to movements of less than 2 Å in atoms of residues of
this region. Movements in side chain atoms of Trp
,
Phe
, and Phe
are the most apparent; these
residues are distant from the active site.
Fig. 5suggests
that the bimane moiety in the modified enzyme may serve as a
fluorescence resonance energy acceptor in the determination of
distances between sites in glutathione S-transferases. Under
nondenaturing conditions, there is substantial reduction of the
intrinsic tryptophan fluorescence by the covalently bound bimane in the
active site. In rat liver glutathione S-transferase 3-3,
there are 4 tryptophan residues/subunit, three of which
(Trp, Trp
, Trp
) are in the range
of 8-14 Å to the bound bimane moiety in the same subunit
(Trp
, 8.2 Å; Trp
, 12.4 Å;
Trp
, 13.8 Å), while the fourth tryptophan as well
as tryptophan residues from the adjacent subunit are more than 25
Å away from the bound bimane moiety in an energy minimized
mB-modified glutathione S-transferase 3-3 model. In
addition, the efficiency of fluorescence energy transfer is inversely
related to the sixth power of the distance between the two
fluorophores(40) . It is, therefore, considered that tryptophan
residues at positions 7, 45, and 214 make the dominant contributions to
the transfer of fluorescence energy to the bimane fluorophore under
nondenaturing conditions. Studies are presently in progress to use this
property to investigate solution conformational properties of the
enzyme.
The data presented in this paper indicate that glutathione S-transferase 3-3 may have two different binding sites for mBBr, one that is identical or overlaps with the CDNB site and a second or alternate site that is independent of the CDNB site. Glutathione S-transferase 3-3 incubated with mBBr in the presence of S-methylglutathione yields a modified enzyme that exhibits only about 6-9% residual activity toward CDNB and bromosulfophthalein but retains full activity toward mBBr, indicating that the site shared with CDNB and bromosulfophthalein is covalently modified by mBBr, while the other independent site remains available. Since the modified enzyme is still catalytically competent toward mBBr, both sites must be very close to the glutathione binding site. There may not be any accessible nucleophiles to react covalently with mBBr in the second site, which would explain why only one site is modified.
Our molecular modeling simulations suggest that after modification
of Tyr by active site-bound mBBr and of Cys
by mBBr approaching from the outside as discussed above, there is
still sufficient room in the xenobiotic binding cavity to bind another
molecule of mBBr. Evidence for the existence of a second xenobiotic
binding site for other glutathione S-transferases has been
obtained previously from equilibrium binding
experiments(41, 42) .
S-Methylglutathione
enhances the rate of reaction between the enzyme and mBBr, probably
through S-methylglutathione-induced conformational change,
which facilitates the binding of mBBr to the enzyme. Fig. 3, inset, showing saturation kinetics for inactivation in the
presence of S-methylglutathione, suggests that mBBr first
binds to the enzyme before covalently modifying the enzyme and causing
inactivation. Comparison of the second order rate constant for reaction
of mBBr with the enzyme alone (0.026 min mM
) with the k
/K
measured in the
presence of S-methylglutathione (0.32 min
mM
) indicates a 12-fold enhancement
of the reactivity of the target site in the presence of the glutathione
derivative. The use of S-methylglutathione in our present
study enables us to reduce the concentration of mBBr needed to obtain
the desired inactivation, thus reducing nonspecific labeling of the
enzyme.
In summary, mBBr modification of rat liver glutathione S-transferase 3-3 occurs initially at the active site
residue Tyr, which leads to loss of catalytic activity
toward CDNB and bromosulfophthalein. Prolonged treatment of the enzyme
with mBBr also results in the modification of Cys
, which
is located in the vicinity of the active site. Glutathione S-transferase 3-3 is shown to have two binding sites for
mBBr, one site that is identical or overlaps with the CDNB site and
another site that is independent of the CDNB site and is also
catalytically active after modification of the first site.