(Received for publication, August 10, 1994; and in revised form, October 31, 1994)
From the
Glutathione transferase P1-1 (EC 2.5.1.18) is a dimeric enzyme composed of identical subunits each containing one binding site for GSH and a second for the co-substrate e.g. 1-chloro-2,4-dinitrobenzene. Steady-state kinetics are strictly hyperbolic toward both these substrates. Replacement of Cys-47 with alanine or serine decreases the affinity for GSH and triggers a positive kinetic cooperativity with respect to this substrate. Hill coefficients were 1.31 and 1.43 for the C47A and C47S mutants. C47A/C101A and C47S/C101S double mutants display lower affinity for GSH and higher Hill coefficients (1.57 and 1.56, respectively) when compared with C47A and C47S single mutants. Conversely, replacement of Cys-101 with alanine or serine does not yield any cooperativity and any marked change of kinetic parameters. Fluorometric experiments gave sigmoidal isothermic GSH binding curves for all the Cys-47 mutants, with Hill coefficients similar to that obtained by the kinetic approach. These data, together with the activation experiments performed in the presence of S-hexylglutathione, suggest that the substitution of Cys-47 yields a dimeric low-affinity enzyme which can be converted to a higher affinity state upon binding of GSH on one subunit. These findings indicate a structural communication between subunits which may be revealed by the lack of a peculiar electrostatic bond between the thiolate form of Cys-47 and the protonated amino group of Lys-54.
The glutathione transferases (EC 2.5.1.18) (GST) ()are a family of detoxicating enzymes able to conjugate the
sulfhydryl function of GSH to a large number of electrophilic
compounds(1, 2, 3, 4, 5, 6) .
The cytosolic GSTs, which have been grouped into four gene-independent
classes, Alpha, Pi, Mu(7) , and Theta(8) , are
homodimeric or heterodimeric proteins composed of subunits of about 25
kDa. Crystal structures of homodimeric GSTs from thee gene classes
(Alpha, Pi, and Mu) have been recently solved (9, 10, 11, 12) and provide useful
data for elucidating the catalytic mechanism. From these structural
studies it is also evident that all these proteins have very similar
tertiary folds. Each subunit can be divided in two domains: a smaller
amino-terminal domain (Domain I) which contains the binding site for
GSH (G-site), and a larger carboxyl-terminal domain (Domain II) which
binds the co-substrate in an hydrophobic cavity or cleft (H-site).
Crystallographic data suggest that, in the dimeric protein, the two
G-sites are structurally independent as well as the two H-sites. The
kinetic independence of the subunits of GSTs has been well established
in studies of a number of rat isoenzymes composed of four different
subunits(13) ; furthermore, steady-state kinetic pathways of
the human placenta GST (class Pi), as well as of the rat liver GST 1-1
(class Alpha), are fully Michaelian and consistent with a rapid
equilibrium random sequential Bi Bi
mechanism(14, 15) . Other isoenzymes such as the rat
GST 3-3 and 3-4 (class Mu) and GST 2-2 (class Alpha) exhibit
non-Michaelian kinetic behavior which would suggest negative
cooperativity, but this pathway has been well fitted with the simplest
steady-state random Bi Bi mechanism(16) , also on the basis of
the strictly hyperbolic binding isotherms of substrates and
products(17) . Thus, it is generally accepted that GSTs lack
kinetic cooperativity or regulatory behavior. In this paper we report
that replacement of a single residue in the Pi isoenzyme triggers a
marked kinetic and ligand binding cooperativity.
During investigations about possible structural roles of Cys-47 and other sulfhydryls in the GST P1-1, we constructed six mutants by different substitutions of Cys-47 and Cys-101, the most reactive cysteines among the four present in each subunit. C47A and C101A mutants of the rat GST P and C47S, C101S mutants of the human GST P1-1 have been previously studied by two different research groups(18, 19) . The kinetic parameters of these mutated enzymes were found approximately similar to those of the wild type but the substitution of Cys-47 lowers the affinity for GSH considerably. No anomalous kinetic behavior has been observed. In this paper we demonstrate that some of these mutated enzymes display positive cooperativity. In particular, the replacement of Cys-47 is crucial in this phenomenon: C47A and C47S mutants and C47A/C101A and C47S/C101S double mutants give similar sigmoidal dependence of the reaction velocity on GSH concentration and homotropic behavior on GSH binding, whereas the C101A and C101S mutants as well as the wild type are noncooperative enzymes.
Cys-47, a conserved
residue in the Pi class isoenzymes, has been the object of many
investigations as it is highly reactive and its chemical modification
inactivates the enzyme possibly by inducing unfavorable conformational
changes for GSH binding or by blocking a structural transition during
catalysis(11) . It is not a structural part of the active site
but it is placed close the G-site(11) . Previously, we
demonstrated that it may form an intrasubunit disulfide link with
Cys-101 (20) leading to an inactive enzyme form. Its sulfhydryl
group has also a very low pK value, and
it is probably linked in ion pair with the protonated group of
Lys-54(21) . Results reported here confirm the key role of this
residue in the activity modulation of GST P1-1.
From this equation a reliable K value
cannot be evaluated because the interaction factor
and K
are not independent
variables.
A sigmoidal curve is obtained from by plotting v versus log [S]. The best fit fulfills V, [S]
, and the Hill
coefficient (n
); (ii) by linear least squares
regression analysis with a linear rate
equation.
In this analysis, V value was obtained by
fitting with .
The dependence of the enzymatic velocity upon CDNB concentration was studied at a constant GSH concentration of 1 mM or 0.5 mM and varying the CDNB concentration from 0.05 mM to 2 mM. Data were analyzed as above or by using Lineweaver-Burk double-reciprocal plots.
Kinetic parameters reported in this paper represent the mean of at least four different experimental sets.
Figure 1:
Homotropic behavior of C47S
mutant. GSH saturation curves of C47S mutant and of wild type at 25
°C were obtained as described under ``Experimental
Procedures.'' Initial velocities are expressed as
A/min. GSH concentrations are expressed as mM.
Each experimental point is the mean of five determinations. Error
bars show the standard error of the mean. The data of C47S mutant
(
) were fitted by nonlinear least squares regression analysis
with . The solid line is the best fit. The dashed line represents the theoretical curve for a
noncooperative GST with the same V
and
[S]
as the mutant. Data of wild type (
)
were fitted by nonlinear least squares regression analysis with assigning n
= 1 (rectangular
hyperbola). The solid line is the best fit. Inset,
Hill plots for C47S mutant (
) and for wild type (
). n
were 1.43 and 1.01,
respectively.
Figure 3:
Activation by S-hexylglutathione.
Experiments were performed at 25 °C as described under
``Experimental Procedures.'' The activity was assayed at
fixed CDNB concentration (1 mM) and at about
0.1[S] GSH concentration.
, wild type at
0.01 mM GSH;
, C47A/C101A mutant at 0.1 mM GSH;
, C101A at 0.03 mM GSH;
, C47S mutant at 0.1
mM GSH.
Figure 4:
Cooperative binding of GSH on the
C47A/C101A mutant. GSH binding was measured by fluorometry as described
under ``Experimental Procedures''. GSH concentrations are
expressed as µM. Data were fitted by nonlinear least
squares regression analysis with . The solid line is the best fit. The dotted line and dashed line are the expected curves for n = 1 (no
cooperativity) and n
= 2 (infinite
cooperativity), respectively.
Figure 5:
Temperature effect on cooperativity.
Kinetic experiments were performed as reported under
``Experimental Procedures'' at the indicated temperatures.
Data were fitted by linear least squares regression analysis with to calculate the Hill coefficients. [S] values for GSH were obtained from . Filled
symbols indicate n
values; unfilled
symbols indicate [S]
values. a,
data for C47S mutant (circles) and for C47S/C101S mutant (squares). b, data for C47A mutant (circles)
and for C47A/C101A mutant (squares).
Two microscopic models are commonly used to explain homotropic behavior in oligomeric enzymes. The first one assumes the enzyme to exist in two conformations, namely R and T, which are in equilibrium and differ in catalytic properties and (or) in substrate affinity (two-state model)(28) . Binding of the substrate shifts the T-R equilibrium toward the most active forms (R) and it originates nonhyperbolic kinetics. In the second model the enzyme may exist in intermediate conformations beside the T and R forms (sequential model) (29) . All the present data referring to the nonhyperbolic steady-state kinetics induced by replacement of Cys-47 in GST P1-1 can be well interpreted by the first model, although the sequential one is equally applicable. It appears that the substitution of Cys-47 yields a low-affinity conformation (T) which can be converted to a higher affinity conformation (R) (similar to that of wild type) upon binding with GSH, as appears in Fig. SI.
Scheme I:
No kinetic cooperativity has been observed in the wild type and in C101A and C101S mutants nor heterotropic and homotropic effects by CDNB in all mutants tested (see Fig. 2b). The independence of the observed cooperativity from CDNB binding or from some kinetic artifacts was also suggested by the cooperative isothermic binding of GSH as determined by fluorescence experiments (see Fig. 4and Table 3), as well as by the activation experiments (see Fig. 3). The higher values of Hill coefficients found for the double mutants compared with the single mutants, as well as the noncooperativity of the C101A and C101S mutants, point for a positive, but not crucial, contribution of the replacement of Cys-101 in the attainment of the R conformation upon GSH binding.
Is there some structural explanation for this induced
cooperativity? As appears from crystallographic data of the enzyme in
complex with s-hexylglutathione(11) , Cys-47 is placed
close the G-site at 10.7 Å from the sulfur atom of the inhibitor
at the end of the irregular helix 2 (residues 35-44). The
crystallographic temperature factors, experiments of limited
proteolysis, and other spectroscopic data indicate that this region is
highly mobile and it undergoes conformational changes upon binding of
GSH(30, 31, 24) . The peculiar ion pair
between Cys-47 and Lys-54 may act as an ``hinge'' which
limits the number of potential conformations. Replacement of the
sulfhydryl moiety of Cys-47 with the undissociated hydroxyl function of
serine or its deletion, as occurs in the C47A mutant, suppresses this
ionic bond. The resulting increased mobility is probably responsible
both for the existence of a GSH low-affinity conformation and for a
possible cooperative split to a high-affinity conformation upon GSH
binding. With this model, the observed temperature dependence of the
Hill coefficients (see Fig. 5) may be explained by assuming that
at low temperatures the C47S and C47A mutants display lower mobility in
this protein portion; the structure of the G-site and its affinity for
GSH become similar to that of the wild type and the cooperativity
disappears. If all the above conclusions are correct, we must find an
induced cooperativity similar to that observed by replacement of Cys-47
when the counterpart in the ion pair with Cys-47, i.e. Lys-54
is substituted with a nonpositive residue. This demonstration will be
given in the subsequent paper in this issue(22) .
Although it was well established the absence of any kinetic cooperativity for GST P1-1, the possibility of a structural communication between the two subunits has been previously suggested to occur in the horse erythrocyte isoenzyme which belongs to the class Pi. In two different papers we stressed the nonequivalence of its subunits in their reaction with sulfhydryl reagents(32, 33) ; possible explanations of this behavior included a nonsymmetrical association of identical subunits or a structural change of the unreacted subunit upon reaction of Cys-47 on the first subunit. In light of the present results, the last possibility appears reasonable.
Conversion of a noncooperative enzyme into a cooperative one by a mutation of a single amino acid residue is an interesting phenomenon; recently, it has been reported that mutation of a specific residue may introduce cooperativity into four previously noncooperative oligomeric enzymes. Some of these enzymes have been known to be evolutionary related to cooperative enzymes. This is the case of the trimeric aspartate transcarbamoylases (EC 2.1.3.2) from Bacillus subtilis and ornithine transcarbamoylase (EC 2.1.3.3) that become cooperative enzymes upon replacement of a single residue at the active site(34, 35) . On the other hand two dimeric enzymes, not related to allosteric enzymes, undergo induced cooperativity; glutathione reductase (EC 1.6.4.2) displays homotropic behavior when a glycine residue, located in the dimer interface, is replaced with a tryptophan residue(36) ; tyrosyl-tRNA synthetase (EC 6.1.1.1.) shows cooperativity upon mutation of Lys-233, a residue involved in the substrate binding (37) .
GST is not related to allosteric proteins. Moreover the mutated residue that triggers cooperativity (Cys-47) is not located in the subunit interface and is not inside the active site. These peculiarities make GST a new and interesting model for induced cooperativity.
From a more general point of view the above findings support the attractive hypothesis that some oligomeric proteins may have the potential to be allosterically controlled. This potential may be expressed by a evolutionary mutation of one or a few amino acids which can transform the oligomeric enzyme into a regulatory enzyme. This did not occur for GSTs; there is no apparent utility of allosteric modulation of activity for an enzyme whose role is to detoxify potentially dangerous chemicals.