Human Glutathione Transferase T2-2 Discloses Some
Evolutionary Strategies for Optimization of Substrate Binding to the
Active Site of Glutathione Transferases*
Anna Maria
Caccuri
§,
Giovanni
Antonini§¶
,
Philip
G.
Board**,
Jack
Flanagan**,
Michael W.
Parker
,
Roberto
Paolesse§§,
Paola
Turella
,
Giorgio
Federici¶¶,
Mario Lo
Bello
, and
Giorgio
Ricci
¶¶
From the
Department of Biology, University of Rome
"Tor Vergata," 00133 Rome, the ¶ Department of Basic and
Applied Biology, University of L'Aquila, 67010 L'Aquila, the
Department of Biochemical Sciences, University of Rome "La
Sapienza," 00185 Rome, Italy, the ** Molecular Genetics Group, John
Curtin School of Medical Research, Australian National University,
Canberra 2601, Australia, 
The Ian
Potter Foundation Protein Crystallography Laboratory, St. Vincent's
Institute of Medical Research, Fitzroy, Victoria 3065, Australia, the
§§ Department of Chemical Science and
Technology, University of Rome "Tor Vergata," 00133 Rome, and the
¶¶ Children's Hospital IRCCS "Bambin Gesù,"
00165 Rome, Italy
Received for publication, April 4, 2000, and in revised form, October 6, 2000
 |
ABSTRACT |
Rapid kinetic, spectroscopic, and potentiometric
studies have been performed on human Theta class glutathione
transferase T2-2 to dissect the mechanism of interaction of this enzyme
with its natural substrate GSH. Theta class glutathione transferases are considered to be older than Alpha, Pi, and Mu classes in the evolutionary pathway. As in the more recently evolved GSTs, the activation of GSH in the human Theta enzyme proceeds by a forced deprotonation of the sulfhydryl group (pKa = 6.1).
The thiol proton is released quantitatively in solution, but above pH
6.5, a protein residue acts as an internal base. Unlike Alpha, Mu, and
Pi class isoenzymes, the GSH-binding mechanism occurs via a simple
bimolecular reaction with kon and
koff values at least hundred times lower
(kon = (2.7 ± 0.8) × 104 M
1
s
1, koff = 36 ± 9 s
1, at 37 °C). Replacement of Arg-107 by
alanine, using site-directed mutagenesis, remarkably increases the
pKa value of the bound GSH and modifies the
substrate binding modality. Y107A mutant enzyme displays a mechanism
and rate constants for GSH binding approaching those of Alpha, Mu, and
Pi isoenzymes. Comparison of available crystallographic data for all
these GSTs reveals an unexpected evolutionary trend in terms of
flexibility, which provides a basis for understanding our experimental results.
 |
INTRODUCTION |
The human cytosolic glutathione transferases
(GSTs,1 EC 2.5.1.18) are
dimeric proteins grouped into at least four gene independent classes,
named Alpha, Mu, Pi, and Theta that differ in amino acid sequence,
co-substrate specificity, and antibody cross-reactivity (for reviews,
see Refs. 1 and 2). Despite an inter-class sequence identity of less
than 25% between Alpha, Mu, and Pi class enzymes and less than 10% to
the Theta class GSTs, the three-dimensional fold of these isoenzymes is
very similar (3-6). Notable differences for the human Theta enzyme
T2-2 are a small and buried active site for GSH and an extra C-terminal
extension of about 40 residues not found in the other classes (6).
Recently a fifth class has been discovered in humans, named Zeta class,
showing a serine residue in the active site and high activity toward
organic hydroperoxides (7). From an evolutionary point of view, it has
been proposed that Alpha, Mu, and Pi class GSTs originated from the
Theta GST by gene duplication (8). In turn, on the basis of sequence identity at the N terminus, the Theta GST should might have arisen from
the ancestral mitochondrial GST Kappa (9). Alternatively, the Theta
class may be only older than Alpha, Pi, and Mu GSTs and have all
diverged from a common ancestor (7). Recently, we have found that human
Alpha, Mu, and Pi class GSTs bind GSH by adopting a very similar
multistep mechanism in which the final Michaelis complex is achieved
after the formation of a weak pre-complex (10, 11). Does such a
mechanism hold for the primitive GSTT2-2? Furthermore, all GSTs
activate the substrate by lowering the pKa value of
GSH at the active site, but the peculiar sulfatase reaction catalyzed
by hGSTT2-2 could not need the thiolate form of GSH (12).
Interestingly, in this old enzyme, Ser-11 replaces the Tyr residue
found in Alpha, Pi, and Mu class GSTs. This aromatic residue contacts
the sulfhydryl group of GSH and stabilizes its ionized form (1).
Recently, Jemth and Mannervik (13, 14) described some kinetic and
binding properties of the rat Theta class GST. They obtained indirect
kinetic indications for a forced deprotonation of GSH at the active
site, and they also proposed Ser-11 as the crucial residue involved in
this activation. We report here, for the human GSTT2-2, direct evidence
for the ionization of GSH at the active site, but we point out the
crucial role of Arg-107 in the binding and activation of the substrate.
This residue contacts the thiol sulfur of GSH, either directly or
through a water molecule (6); replacement of Arg-107 by Ala not only alters the pKa value of the bound GSH but even
changes the binding mechanism of this enzyme. Interestingly, some of
the binding properties of the old enzyme seem to be related to the rigidity of selected regions of the protein, indicating a possible evolutionary target in terms of flexibility for the GST superfamily.
 |
EXPERIMENTAL PROCEDURES |
Reagents and Enzyme Preparation--
GSH and
S-hexylglutathione are Sigma products; sodium 1-menaphthyl
sulfate (Msu) was prepared as described by Clapp and Young (15).
His-tagged recombinant GST2-2 and R107A mutants were expressed in
Escherichia coli and purified using immobilized metal ion
chromatography on a nickel-nitrilotriacetic acid matrix (Qiagen) as
described previously (12, 16).
GSH Binding Experiments--
The intrinsic fluorescence of
hGSTT2-2 was measured in a single photon counting spectrofluorometer
(Fluoromax, S.A. Instrument, Paris, France) with a sample holder at
25 °C. Excitation was at 280 nm, and emission was at 335 nm. In a
typical experiment, fluorescence intensity was measured before and
after the addition of suitable amounts of GSH (from 0.02 to 8 mM) to 1.5 µM hGSTT2-2 in 0.1 M potassium phosphate buffer, pH 7.0. Experimental data were corrected both for dilution and for inner filter effects and fit to
Equation 1,
|
(Eq. 1)
|
where F0 is the protein fluorescence in
the absence of GSH; FL is the protein fluorescence
in the presence of a given amount of GSH; Fmax
is the protein fluorescence at saturating GSH concentrations, and
nH is the Hill coefficient.
Spectroscopic Evidence for GSH Ionization--
Difference
spectra of GSH thiolate bound to both native and the R107A mutant of
hGSTT2-2 were obtained with a Kontron double-beam Uvikon 940 spectrophotometer thermostated at 25 °C. In a typical experiment 1 mM GSH was added to the enzyme (15 µM active
sites) in a suitable buffer. From the resulting spectrum, the
contributions from free GSH and free enzyme were subtracted. The amount
of thiolate was obtained by assuming an
240 nm of 5,000 M
1 cm
1.
The pH dependence of the GSH ionization was obtained with the following
buffers (0.01 M): sodium acetate buffer, pH 5.5, and potassium phosphate buffers between pH 6.0 and pH 8.0. pKa values were obtained by fitting the data to
Equation 2.
|
(Eq. 2)
|
Potentiometric Experiments--
Thiol proton extrusion was
detected at 25 °C as reported previously (10). In a typical
experiment, performed under a N2 atmosphere, a GSH solution
(10 mM in 0.1 M NaCl) was titrated to a fixed
pH with 0.1 M NaOH and mixed with the same volume of GSTT2-2 (4 mg/ml in 0.1 M NaCl) at exactly the same pH.
Quantitation of the released proton was obtained by suitable addition
of 10 mM NaOH up to the initial pH value. A blank was
performed at each pH, by replacing GSH with
S-hexylglutathione (4 mM in 0.1 M
NaCl). pKa values were obtained by fitting the data
to Equation 3,
|
(Eq. 3)
|
Stopped-flow Experiments--
Rapid kinetic experiments were
performed on an Applied Photophysics Kinetic spectrometer stopped-flow
instrument equipped with a thermostated 1-cm light path observation
chamber. Kinetics of the binding of GSH to wild-type and R107A mutant
were studied at 37 °C, by rapid mixing of enzyme (95 µM) and different amounts of GSH (from 0.25 to 12 mM) in carbonate/phosphate/acetate (50:50:50 µM) buffer, pH 7.0. Binding of GSH was monitored by
following the increase of the intrinsic fluorescence of the protein.
Experimental traces were fit to a single exponential decay, and
pseudo-first order kinetic constants were calculated at different GSH concentrations.
Binding of GSH as Function of pH--
Binding of GSH as function
of pH was performed at 37 °C. Wild-type or R107A mutant (95 µM) in carbonate/phosphate/acetate (50:50:50
µM) buffer were rapidly mixed with the same volume of GSH
(2.0 mM) dissolved in the same buffer. Experiments were
done at different pH values between pH 5.0 and pH 9.0. Binding of GSH was monitored by following the increase of the intrinsic fluorescence of the protein. Experimental traces were fit to a single exponential decay, and pseudo-first order kinetic constants were calculated.
 |
RESULTS |
Binding of GSH to hGSTT2-2--
Isothermic binding of GSH to the
G-site of hGSTT2-2 has been studied at 25 °C by using the
perturbation of the intrinsic fluorescence of the protein upon addition
of different GSH concentrations. In the native enzyme, the interaction
of the substrate with the active site causes an increase of the
tryptophan fluorescence, and this is a peculiar finding as other GST
isoenzymes display a fluorescence quenching on GSH binding. Binding of
GSH is hyperbolic (nH = 1.0) with an
[S]0.5 = Kd value of 0.8 ± 0.2 mM (Fig. 1A). This
value is at least four times higher than those found for the more
recently evolved Alpha, Pi, and Mu class GSTs. This poor affinity for
GSH is also reflected by the lack of interaction of this GST with the
classical GSH affinity matrix (12).

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Fig. 1.
Binding (A) and GSH thiolate
formation in hGSTT2-2 and in the R107A mutant
(B). A, GSH binding was measured by
fluorometry as described under "Experimental Procedures," and data
were fit to Equation 1. B, spectra of GS in
the binary complex with hGSTT2-2 ( ) and with R107A mutant ( ),
were obtained under different pH conditions with 15 µM
active sites and 1 mM GSH (see "Experimental
Procedures"). The amount of thiolate formed at the G-site was
calculated by assuming an 240 nm of 5000 M 1 cm 1.
The solid lines are the best fit of the data to Equation 2.
Inset, difference spectrum of the hGSTT2-2-GSH binary
complex in 0.01 M potassium phosphate buffer, pH 7.5. The
S.E. for each point does not exceed 5 and 9% for hGSTT2-2 and R107A
mutant, respectively.
|
|
Evidence of Thiolate Formation in hGSTT2-2 and in R107A
Mutant--
Direct demonstration of GSH thiolate formation at the
active sites of hGSTT2-2 and of R107A mutant and their dependence on pH
have been obtained by differential UV spectroscopy. Fig. 1B, inset, shows a typical thiolate absorption band centered at
240 nm obtained at pH 7.5 by the differential UV spectrum of hGSTT2-2 in the presence of nonsaturating GSH concentration (1 mM).
Similar thiolate band is obtained with the R107A mutant. Higher GSH
concentrations cannot be used because of the large spectral
contribution due to the spontaneous ionization of GSH at alkaline pH
values. By assuming an
240 nm of 5,000 M
1 cm
1
for the ionized GSH, the limiting value at alkaline pH is 0.48 GS
equivalents per hGSTT2-2 active sites when the active
site occupancy is about 55%. The dependence, at pH 7.0, of the GSH
thiolate band at 240 nm on GSH concentration (from 0.1 to 1 mM) follows a hyperbolic behavior that overlaps the GSH
binding experiments (see Fig. 1A) (data not shown). It
follows that about 0.9 GS
/active sites are formed in the
native enzyme at 25 °C under saturating substrate. The pH dependence
of the spectral perturbation at 240 nm gives an apparent
pKa value for the bound GSH of 6.15 ± 0.10 (Fig. 1B), close to the pKa values found
for Alpha, Mu, and Pi class GSTs (10, 11). Mutation of Arg-107
remarkably decreases the deprotonation of the substrate, and a
pKa = 7.8 ± 0.2 for the thiol group of the
bound GSH is now obtained (Fig. 1B), a value 1.6 pH units
higher than that calculated for the native enzyme.
Proton Release of upon GSH Binding to hGSTT2-2--
The fate of
the proton produced from the GSH ionization has been investigated in
hGSTT2-2 by a potentiometric approach as described under
"Experimental Procedures." In a typical experiment (pH 6.5), a
nearly saturating GSH solution (5 mM final concentration) was mixed with hGSTT2-2 (73 µM final concentration), both
solutions in the absence of any buffer, and titrated to the same pH
value. After mixing, a rapid decrease of pH was observed. No pH changes have been found when the GSH analogue S-hexylglutathione
replaces GSH. These experiments show that in hGSTT2-2, protons are
released upon GSH binding and that they come from the sulfhydryl group of GSH. Back titration with dilute NaOH allows an estimation of the
amount of the released proton. The pH dependence of this event shows a
bell-shaped trend (Fig. 2). It is evident
that under alkaline conditions at least one protein residue acts as an
internal base for proton neutralization. By fixing the
pKa1 = 6.15 (the value obtained for GSH
ionization by differential UV spectroscopy), the best fit of the
experimental data gives an apparent pKa2 of 7.32 ± 0.03 for the unknown protein base.

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Fig. 2.
pH dependence of the thiol proton release in
hGSTT2-2. The concentration of the released thiol proton
([H+]released) was calculated as described
under "Experimental Procedures" and normalized to the quantity of
active sites. Lines are the best fit of data to Equation 3.
, human GSTT2-2; , pH dependence of thiol proton release
previously reported for the Delta GST (17). The S.E. for each point
does not exceed 8%.
|
|
Kinetics of GSH Binding to hGSTT2-2 and to R107A
Mutant--
Kinetics of GSH binding was studied at pH 7.0 and
37 °C, by following the increase of protein fluorescence in the
milliseconds time scale by a stopped-flow apparatus. The experimental
traces, obtained after rapid mixing of enzyme with increasing GSH
concentrations, were well described by single exponential curves from
which the apparent first order rate constants
(kobs) have been calculated. The native enzyme
shows kobs values (65 s
1 at 1 mM GSH and 37 °C) at
least 10 times lower than those found for the Alpha, Mu, and Pi class
GSTs at the same GSH concentration but at 5 °C (10, 11).
Furthermore, unlike that observed in the more recently evolved GSTs,
kobs values for hGSTT2-2 follow a linear
dependence on GSH concentration within the large range of 0.12-6
mM (Fig. 3A).
These data are well described by Scheme I, which shows the binding
event as a simple bimolecular interaction to give the Michaelis complex
E-GSH.

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Fig. 3.
Kinetics of GSH binding to hGSTT2-2 and to
R107A mutant. The observed rate constants
(kobs) for binding of GSH to the enzymes were
obtained by stopped-flow experiments performed at 37 °C and pH 7.0. With hGSTT2-2, the kobs values show a linear
dependence on GSH concentration (A, ) and linear
regression analysis gives a kon = (2.7 ± 0.8) × 104 M 1
s 1 and a koff = 36 ± 9 s 1 for the equilibrium between
GSH and enzyme. With R107A mutant, The kobs show
a nonlinear dependence on GSH concentration (A, ). The
solid line is the best fit of the experimental data to
Scheme II. The pH dependence of kobs is shown in
B for hGSTT2-2 ( ) and for R107A mutant ( ). Experiments
were performed at fixed GSH concentration (1 mM) and at
different pH values between pH 5.0 and 9.0. The S.E. for each point
does not exceed 4%.
|
|
Linear regression analysis gives a kon = (2.7 ± 0.8) × 104
M
1 s
1
and a koff = 36 ± 9 s
1. Thus, a dissociation constant
(Kd) of about 1.3 mM for the
E-GSH binary complex formation has been calculated at 37 °C (Fig. 3A and Table
I). As kon is far
from the value expected for a diffusion limited process, a rapid
equilibrium between at least two G-site conformations is likely. Only
the less populated conformation should be competent for a proper
interaction with GSH. The mutant enzyme always displays
kobs values higher (5-10-fold) than those
observed for the native enzyme at the same temperature and at the same
GSH concentration (Fig. 3A). For example, at 1 mM GSH, kobs is about 500 s
1 in the R107A mutant which is 65 s
1 in the native enzyme. In addition, the
dependence of the observed rate constants on GSH concentration is not
linear, but now it follows a hyperbolic behavior. This kinetic trend is
similar to that found for Alpha, Mu, and Pi class GSTs (10, 11), and it
is diagnostic for a multistep binding mechanism (see Scheme II).
The proposed minimal scheme describes a first fast interaction of
GSH with the enzyme to give a weak pre-complex which is slowly
converted into the final Michaelis complex. Only this final complex is
responsible for the fluorescence perturbation at 340 nm. Nonlinear
fitting of all experimental data to Scheme II gives a
kon
1 × 105
M
1 s
1
(the angular coefficient of the tangent to the hyperbola at the lowest
GSH concentrations) and a koff
140 s
1. These values are about 4-fold higher than
those calculated for the native enzyme. The resulting dissociation
constant for the pre-complex
(koff/kon) is 1.4 mM. The microscopic rate constants for the conversion of
this pre-complex into the final Michaelis complex are
k2 = 741 ± 50 s
1
and k
2 = 215 ± 30 s
1. The overall dissociation constant
((koff/kon) × (k
2/k2)) is 0.4 mM, a value lower than that observed for the native enzyme (see Table I).
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Table I
Microscopic rate constants for the binding of GSH to hGSTT2-2 and
R107A mutant
Experiments were performed at 37 °C as described under
"Experimental Procedures." Definitions of microscopic rate
constants are given in Schemes I and II for the wild-type and R107A
mutant, respectively.
|
|
pH Dependence of the Rate of GSH Binding--
The effect of pH on
the rate of GSH binding has also been analyzed. In hGSTT2-2, the
kobs increases at low pH values, and at pH 5.0 (kobs = 110 s
1) it is about
4-fold higher than that observed at pH 9.0 (kobs = 29 s
1) (Fig. 3B). This suggests
that the protonation of one or more protein residues facilitates a
correct interaction with the substrate. In R107A mutant, GSH binds with
kobs values higher than that shown for the wild
type at the same pH value but with a similar pH dependence (Fig.
3B). Thus one or more residues, but not Arg-107, are
involved in the observed facilitation of GSH interaction at acidic pH values.
 |
DISCUSSION |
The first observation coming from the present data is that
hGSTT2-2 is able to activate GSH with an efficiency very similar to
that shown by the more recently evolved GSTs. In fact, the apparent
pKa for the sulfhydryl group of the bound GSH is
6.15, a value in the range of those observed for Alpha, Mu, and Pi
class GSTs (pKa = 6.0-6.8). This value, obtained by
a direct spectroscopic determination of the thiolate ion, is close to
that calculated by kinetic experiments for rat T2-2 enzyme (13). Thus,
these findings indicate that the substrate activation is a property
acquired early by GSTs and strictly conserved during evolution. The
mechanisms for activation and stabilization of the sulfhydryl atom,
however, could be different among GSTs; the thiolate is hydrogen-bonded
to a Tyr residue in the more recently evolved GSTs (1), whereas a
similar role is played by a serine residue in the insect Delta class
GST previously classified as a Theta-like GST (17). Even hGSTT2-2 shows
a Ser residue and not a Tyr, within hydrogen bonding of the GSH
sulfhydryl group (6). The substitution of Ser-11 by Ala in the rat
GSTT2-2 seems to cause an increase of the GSH pKa of
1.3 pH units (14). However, we noted that in the human Theta GSTT2-2,
Arg-107 is in hydrogen bonding distance of the main chain carbonyl of
the
-glutamyl moiety of GSH and forms an interaction with the thiol sulfur of GSH either directly or through a water molecule (6). Subsequent mutagenesis and modeling studies suggested this residue is
involved in the sulfatase reaction and in electrostatic substrate recognition (16). The present data indicate that mutation of Arg-107
has a remarkable negative effect on the deprotonation of the substrate
GSH. In R107A mutant, the apparent pKa of the bound
GSH shifts from pH 6.1 to pH 7.8. Arg-107 could act both as a
counterion to promote ionization of the GSH thiolate and then stabilize
the thiolate by direction interaction. This is not the first case of
arginine residues being implicated in GSH activation. In hGSTA1-1
(Alpha class), Arg-15 is within hydrogen bonding distance of the GSH
thiol (18), and its activation/binding role was subsequently confirmed
by mutagenesis (19). In hGSTM2-2 kinetic data are consistent with
Arg-107 playing a role in promoting ionization and binding of GSH (20).
In conclusion, on the basis of our data on the human enzyme and of
those on the rat GSTT2-2 (13, 14), it is likely that GSH activation is
achieved by the synergistic action of at least two residues, Arg-107
and Ser-11. As for the Alpha, Mu, and Pi GSTs, this old enzyme also
extrudes quantitatively the thiol proton of GSH from the active site
into the surrounding solution but only up to pH 6.5. Above this value, a protein residue acts as a base in capturing the thiol proton (see
Fig. 2). The apparent pKa of this internal base is
7.3, a value that suggests the involvement of a histidine residue, possibly His-40, which is located close to the bound GSH. A nitrogen atom of the imidazole ring of His-40 is in van der Waals contact of the
glycyl moiety of GSH. The capture of the thiol proton at high pH values
has been also observed in the Delta GST (17). In that case His-38
and/or His-50 are probably involved. The ability to release
quantitatively the thiol proton in solution at any pH value is probably
an evolutionary advantage reached by the Alpha, Mu, and Pi class GSTs;
during the enzymatic turnover, the proton neutralized by the protein
residue in hGSTT2-2 must be released before a new productive cycle can
start, and this step may limit the overall velocity.
A second aspect we analyzed is the thermodynamic and kinetic efficiency
of substrate binding to hGSTT2-2. This old enzyme shows a low affinity
for GSH as suggested by an apparent Kd of 0.8 mM, a value at least four times higher than that found in
the more recently evolved GSTs. It appears that Alpha, Mu, and Pi GSTs
are under an evolutionary pressure in the direction of lower
Kd values. This trend is distinctive for enzymes that exhibit kcat/Km ratios
far from the diffusion control limit (108-1010
M
1 s
1)
(21). Actually, all GSTs are distant from a perfectly evolved catalyst,
and hGSTT2-2 shows a specificity constant for GSH of only
102 M
1
s
1. Moreover, a first enzyme on a metabolic
pathway (like hexokinase in the glucose metabolism) often evolves
toward a lower Km (or Kd) value
to prevent accumulation of intermediates; this could be the case of GST
as the starting enzyme in the mercapturic acid formation.
From a kinetic point of view, the observed rate constants for binding
of GSH to the G-site (at 37 °C) are remarkably lower (about 10 times) than those observed in the Alpha, Mu, and Pi GSTs (at 5 °C)
(10, 11). Above all, the most striking peculiarity of the Theta class
enzyme is the linear dependence of kobs on GSH
concentration which is consistent with a single step binding mechanism,
i.e. the Michaelis complex is formed by a bimolecular encounter between GSH and enzyme. Is it possible to elucidate the
evolutionary pathway GSTs have utilized to optimize their interaction
with the substrate? Our data suggest a shifting from a single step
binding mechanism to a multistep binding process, such as observed in
Alpha, Mu, and Pi GSTs (10, 11). These younger GSTs are able to
interact with GSH through a weak pre-complex, followed by at least one
isomerization step that results in a more rapid attainment of the
binding equilibrium. This strategy is reminiscent of that used by many
enzymes in catalysis where a single specific reaction is normally
subdivided into a number of chemical steps with lower activation
energies. From a structural point of view, it is reasonable to propose
that deletion of the extra C-terminal segment, which mostly obscures
the G-site of the human Theta class (6), should facilitate the GSH
interaction. Furthermore, other important factors must be considered
that refer to the dynamics of this enzyme. A plot of the
crystallographic B-factors along the polypeptide chain can give an
indication of the relative flexibility of a protein portion compared
with other regions. As shown in Fig. 4
the Alpha, Mu, and Pi GSTs display a similar and well defined
flexibility pattern. Several "hot" regions with high mobility can
be identified (helix 2 and its flanking regions, C-terminal segment of
helix 4, loop between helices 4 and 5, and N-terminal segment of helix
5) separated by a number "cold" segments. The hGSTT2-2 enzyme shows
a completely different flexibility as no distinctive hot and
cold regions can be defined (the noise in T2-2 plot is due to the
limited resolution of the crystal structure) (Fig. 4). It appears that
GSTs have utilized flexibility in terms of an evolutionary progression. Flexibility explains some of the behavior of the R107A mutant. Arg-107
forms a salt bridge with Asp-104, and its replacement by Ala probably
increases the structural flexibility of the enzyme. This is a plausible
explanation for the remarkable increase of the rate of the GSH binding
in the R107A mutant. In addition, replacement of Arg-107 changes the
binding event from a single step to a multistep mechanism (see Fig.
3A), as observed in the more recently evolved GSTs. In other
words, it appears that this specific substitution in the Theta class
enzyme produces an improvement of the kinetic efficiency of GSH
binding, and the mutant enzyme now resembles the behavior of the more
recently evolved GSTs.

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Fig. 4.
Mobility profiles of
human Alpha, Mu, Pi, and Theta class GSTs. The mobility
profiles are derived from the crystallographic temperature factors of
A1-1 (1GSF) (A), M2-2 (1HNA) (B), P1-1 (6GSS)
(C), and T2-2 (1LJR) (D) GST isoenzymes.
|
|
 |
ACKNOWLEDGEMENT |
We thank Prof. M. Coletta for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by the Italian Ministry of
University and Scientific and Technological Research Grant MURST (60%) and MURST PRIN (40%) (to G. R.) and by National Research Council of
Italy Grant (Target Project on Biotechnology) (to G. R. and G. A.).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.
§
Both authors equally contributed to this work.

To whom correspondence should be addressed: Dept. of
Biology, University of Rome "Tor Vergata," Viale della Ricerca
Scientifica, 00133 Rome, Italy. Tel.: 39 6 72594379; Fax: 39 6 2025450; E-mail: riccig@uniroma2.it.
Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M002819200
 |
ABBREVIATIONS |
The abbreviations used are:
GST, glutathione
transferase;
Msu, 1-menaphthyl sulfate.
 |
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