(Received for publication, August 10, 1994; and in revised form, October 31, 1994)
From the
In the human placental glutathione transferase, Cys-47
possesses, at physiological pH values, a pKvalue of 4.2 and may exist as an ion pair with the protonated
-amino group of Lys-54. Using site-directed mutagenesis we
investigate spectral, kinetic, and structural properties of Cys-47 and
Lys-54 mutants. The results shown indicate that the thiolate ion
detected at 229 nm should be assigned exclusively to Cys-47. The
contribution of Lys-54 to the activation of Cys-47 is assessed by the
spectral properties of the K54A mutant enzyme. The induced
cooperativity toward glutathione, as a consequence of mutation of
Lys-54 to alanine, clearly parallels that observed for the Cys-47
mutant enzymes (see the preceding paper (Ricci, G., Lo Bello, M.,
Caccuri, A. M., Pastore, A., Nuccetelli, M., Parker, M. W., and
Federici, G.(1995) J. Biol. Chem. 270, 1243-1248) and
points out the importance of this electrostatic interaction in shaping
the correct spatial arrangement for the binding of glutathione and in
anchoring the flexible helix
2. When this ion pair is disrupted,
by mutation of either residue, the flexibility of this region could be
greatly increased, causing helix
2 to come in contact with the
other subunit and generating a structural communication, which is the
basis of the observed cooperativity.
The glutathione transferases (EC 2.5.1.18) (GST) ()are a family of enzymes involved in the mechanism of
cellular detoxification. They catalyze the nucleophilic attack of
glutathione on the electrophilic center of a number of toxic compounds
and xenobiotics(1) . The cytosolic enzymes have been grouped
into four species-independent classes: Alpha, Mu, Pi, and Theta, on the
basis of N-terminal sequence, substrate specificity and immunological
properties(2, 3) .
Human placental glutathione
transferase (Class Pi) (GST P1-1)(4) , a homodimeric protein of
about 46 kDa, has been extensively studied in different laboratories
because of the clinical interest in it as a potential marker during
chemical carcinogenesis (5, 6) and its potential role
in the mechanism of cellular multidrug resistance against a number of
antineoplastic agents(7, 8, 9) . Although a
number of amino acid residues involved in the binding of GSH have been
identified by crystallographic analysis (10) and site-directed
mutagenesis studies (11, 12, 13) , the
catalytic mechanism is not completely clarified. Moreover, the key
structural role of some residues is still unknown. The function of
cysteinyl residues has been extensively investigated (14, 15) , leading to the conclusion that none of
these residues is involved in the catalysis. However, the surprising
results that mutation of Cys-47 in alanine or serine induces a positive
cooperativity between the two subunits (see the preceding
paper(29) ) shed more light on the importance of this residue
and prompted us to investigate further its structural role. We
previously suggested that this residue exists as an ion pair with the
protonated -amino group of Lys-54 (16) and its thiolate
form is characterized by a sharp UV absorption spectrum centered at 229
nm. However, spectral and kinetic experiments together with
electrostatic calculations gave us only indirect evidence of the ion
pair existence. In this paper we investigate, by site-directed
mutagenesis, the role of both these residues providing answers to
questions such as: 1) given the fact that there are two reactive
cysteines (Cys-47 and Cys-101) can the spectral band centered at 229 nm
be assigned exclusively to Cys-47? 2) Is Lys-54 the only positive
charge responsible for the activation of Cys-47? 3) If the
cooperativity induced by mutation of Cys-47 is a consequence of
disruption of the ion pair, does the mutation of Lys-54 (the counter
part of the ion pair) also causes positive cooperativity? To this aim
we have replaced Lys-54 with Ala to neutralize the side chain positive
charge of this residue. This mutant enzyme was expressed in Escherichia coli, purified and its kinetic and spectral
properties investigated by comparison with the wild type and the
cysteine mutant enzymes. The results shown in this paper suggest that
the thiolate spectral band should be assigned exclusively to Cys-47 and
that mutation of Lys-54 causes a remarkable decrease of the thiolate
spectral band and also induces positive cooperativity. Limited
proteolysis experiments with trypsin were also carried out on all these
mutant enzymes to probe for conformational changes upon replacement of
either of the residues of this ion pair. Finally, heat inactivation
studies were performed on the same mutants to assess whether the
induced cooperativity was achieved at the expense of some loss of
thermal stability of the protein.
Wild type and mutant enzymes were purified by affinity chromatography on immobilized glutathione(24) .
After affinity purification the wild type and the mutant enzymes were all homogeneous as judged by reverse-phase HPLC and SDS-PAGE. The retention time in the HPLC system and the apparent subunit molecular weight (about 23 kDa) were similar to those of the wild type enzyme. The kinetic parameters of the K54A mutant enzyme are reported in Table 1.
Figure 1: UV difference spectra of wild type and cysteine-mutant enzymes after reaction with bromopyruvate. 2.2 µM wild type (4.4 µM Cys-47) or cysteine mutant enzymes in 0.1 M potassium phosphate buffer, pH 7.0, were placed in the sample and the reference cuvettes and 17.6 µM (final concentration) bromopyruvate was added in the sample compartment. A, wild type (curves a, b, and c: time 0, 1, and 15 min, respectively); B, C101A mutant (curves a, b, and c: time 0, 1, and 20 min, respectively); C, C47A mutant (curves a, b, and c: time 0, 4, and 120 min, respectively); D, C47A/C101A mutant (curves a, b, and c: time 0, 3, and 120 min, respectively). Inset, time course of inactivation.
Figure 2:
pH dependence of the apparent molar
extinction of the spectral band at 229 nm. 2.2 µM of wild
type or K54A mutant enzymes were placed in the sample and reference
cuvettes in the presence of 0.1 M buffer at suitable pH
values. The reaction was started by addition of 8.8 µM bromopyruvate in the sample. The apparent values were
calculated at the end of the reaction, when both the absorbance at 229
nm, and the activity values became constant.
, wild type;
,
K54A mutant enzyme.
Figure 3:
Homotropic behavior of K54A mutant enzyme.
GSH saturation curve of the K54A mutant enzyme at 25 °C was
obtained as described under ``Experimental Procedures.''
Initial velocities are expressed as A/min. GSH
concentration is expressed as mM. The data were fitted by
nonlinear least squares regression analysis with Equation 1 previously
described(29) . The line of best fit is shown. Inset,
Hill plot of data. n
value was
1.4.
Figure 4:
Thermal
stability of the wild type and mutant enzymes. The enzyme was subjected
to heat inactivation studies as described under ``Experimental
Procedures.'' The remaining activity was assayed after 10 min of
incubation at different temperature values. , wild type;
,
C47A mutant; asterisk, C101A mutant;
, C47A/C101A
mutant;
, C47S mutant;
, C47S/C101S mutant;
, K54A
mutant.
Chemical modification studies on GST P1-1 using thiol
reagents suggested that Cys-47 is the likely target of this reaction
causing a fast inactivation
process(36, 37, 38) , even though this
residue is not involved in the catalytic
mechanism(14, 15) . The possible reason for this
finding may rely on the particular position of this residue which is
situated at the end of the flexible helix 2, close to the G-site.
Crystallographic analysis of the GST P1-1, in complex with S-hexylglutathione, reveals that Cys-47 is located on the
surface with its thiol group pointing into a small hydrophobic pocket,
formed by some main chain atoms of Lys-44 and Gln-51 and the side chain
atoms of Trp-38 and Leu-52. Since the same residues are involved, on
the opposite side, in the binding of GSH one could explain why chemical
modification of Cys-47 disturbs the binding of GSH, whereas the
presence of this substrate renders Cys-47 less accessible to the
solvent(39) . Thus, Cys-47 may act as a molecular switch for
different conformations, depending whether GSH or thiol-reagents bind
first to the enzyme. A very similar behavior has been observed for
Cys-289, a residue close to
-Glu-Cys site in the glutathione
synthetase from E. coli(40) . Thus, the role played by
Cys-47 in GST P1-1 could be also of more general interest for different
proteins.
Using site-directed mutagenesis we have demonstrated that
the thiolate ion detected at 229 nm should be assigned exclusively to
Cys-47 (see Fig. 1) despite of the fact that Cys-101 displays
high reactivity and its modification inactivates the enzyme. The
existence of the ion pair with Lys-54 is suggested from the substantial
decrease of value at 229 nm and the shift in the pK
value from 4.2 to 5.3 for the thiol group, observed in the K54A
mutant enzyme (see Fig. 2). These results need to consider other
important electrostatic interactions in lowering the pK
value of Cys-47, in the case of K54A mutant. One possibility is
that the flexible helix
2, where Cys-47 is located, may undergo
an increased motion by approaching the thiol group to on other positive
charge. As an example, Lys-44 is 7.9 Å from the sulfur atom of
Cys-47 in the three-dimensional structure complexed with S-hexylglutathione, but this distance could be substantially
shortened during this motion. Therefore, Lys-44 could be the putative
residue which contributes to the formation of fluctuating ion pair, in
the absence of Lys-54. The hypothesis suggesting structural changes is
corroborated by experiments employing limited proteolysis which
demonstrate that replacement of Lys-54 with alanine leads to an
increased proteolytic susceptibility.
The homotropic behavior found
for the K54A mutant enzyme (see Fig. 3) clearly parallels the
positive cooperativity shown by the Cys-47 mutant enzymes. It is
evident that the electrostatic interaction
(Cys-47-Lys-54
) is important in
shaping the correct spatial arrangement for the binding of GSH to the
active site and in anchoring the flexible helix
2. When this ion
pair is disrupted, by mutation of either residue, the flexibility of
this region could be greatly increased, causing helix
2 to come
in contact with the other subunit and generating a structural
communication, which is the basis of the observed cooperativity. To
determine if this novel phenomenon leads to some change in the thermal
stability of GST P1-1, heat inactivation experiments were carried out
with the wild type and the mutant enzymes. The results clearly indicate
that the cooperativity acquired by mutation of Cys-47 residue does not
change the thermal stability properties shown by the wild type enzyme.
In contrast, mutation of Lys-54 causes a lose of catalytic activity at
a substantially lower temperature (see Fig. 4). It may therefore
be concluded that while the kinetic parameters proteolytic experiments,
and induced cooperativity of Cys-47 and Lys-54 mutant enzymes indicate
a rearrangement of the helix
2 due to disruption of the ion pair,
thermal stability properties are not related to the existence of this
electrostatic interaction and may reflect an additional structural role
for Lys-54.
As an hypothetical structural explanation, we can speculate from inspection of the three-dimensional structure of GST P1-1 (see Fig. 5) as follows: mutation of Cys-47 leads to structural perturbation in the G-site resulting in a reduced affinity for glutathione. The perturbation is also transmitted to the other monomer, possibly via helix 4 which consists of residues involved in subunit contacts (Asp-98, Asp-94, Met-91) and residues implicated in the binding and catalysis of substrate (Tyr-108, Ile-104, Cys-101, Asp-98). Residues Tyr-49 and Gln-64 are also implicated in the transmission, because they are located close to Cys-47 and are also involved in subunit contacts with helix 4 of the other monomer. The binding of glutathione appears sufficient to correct the perturbations in helix 4 due to the Cys-47 mutation, leading to return of the high affinity binding site in the second monomer. Crystallographic studies of the Cys-47 and Lys-54 mutant enzymes will clarify structural transitions coupled with local structural changes during the allosteric transitions.
Figure 5: Ribbon representation of the human glutathione transferase P1-1 showing potential residues involved in the observed positive cooperativity (see text). The view is taken down the 2-fold axis of the dimer molecule. The figure is based on the crystal structure of the enzyme in complex with S-hexylglutathione (10) (the inhibitor is denoted by hexyl and GSH in the figure) and was produced by the computer program MOLSCRIPT(19) .