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INTRODUCTION |
Human glutathione transferase T2-2 (hGSTT2-2, Theta class) is
thought to be directly descended from the ancestral precursor of the
more recently evolved GSTs1
found widely expressed in humans (1, 2). All members of the GST
superfamily catalyze the nucleophilic addition of GSH to a variety of
electrophilic compounds, many with carcinogenic and toxic properties,
thus favoring their excretion (3, 4). Human cytosolic GSTs have been
grouped into at least five gene-independent classes named Alpha, Pi,
Mu, Theta, and Zeta, which differ in their co-substrate and inhibitor
specificity as well as in antibody reactivity (3-5). Despite low
sequence homology, all of these isoenzymes have very similar
three-dimensional structures and very similar topography of the G-site
(6-9). The hGSTT2-2 is a homodimeric protein characterized by an
additional 40 odd residues at the C terminus and by a peculiar
sulfatase reaction (8) not found in the more recently evolved Alpha,
Pi, and Mu GSTs. The C-terminal extension, comprising two helices
connected by a long loop, completely buries the substrate-binding
pocket and occludes most of the GSH-binding site. By virtue of the
extension being wedged into the active site, mobile regions found in
other GSTs such as helices
2 and
4 are no longer very flexible.
In the accompanying paper (10), we have reported that hGSTT2-2 binds
GSH with a mechanism different and less efficient than that observed in
the other transferases (11, 12), suggesting an evolutionary strategy
adopted by the GST family to optimize this process. In this paper we
dissect the catalytic mechanism of hGSTT2-2 by means of steady state
and pre-steady state kinetic experiments and site-directed mutagenesis.
Most of the kinetic studies have been carried out using 1-menaphthyl
sulfate (Msu) as co-substrate. A few experiments utilized
1-chloro-2,4-dinitrobenzene (CDNB), one of the best co-substrates for
the Alpha, Pi, and Mu GSTs but which was previously considered not to
be a substrate for the Theta enzyme (13). The crystallographic studies
demonstrated that the crystals were catalytically active in that they
could convert the sulfatase substrate, Msu, into the corresponding GSH conjugate with cleavage of the sulfate group. Surprisingly, there was
no evidence that the C-terminal extension moves away from the active
site, and it was suggested that a narrow tunnel might widen to allow
the passage of substrates and products between the active site and the
surrounding solvent. In accordance with these data, all kinetics
reported here depict a somewhat primordial catalysis that is
rate-limited by the product release in the case of Msu and by a less
than optimal chemical step in the case of CDNB. Recently, two kinetic
studies on the rat T2-2 enzyme have been published (14, 15). In those
papers, a hysteretic mechanism has been proposed that involves at least
three or four different interconverting enzyme conformers. A different
kinetic scenario is described here for the human isoenzyme that
displays a peculiar half-site catalysis due to a slow or absent
interconversion between two catalytically distinct enzyme populations
(or active sites).
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EXPERIMENTAL PROCEDURES |
Reagents and Enzyme Preparation--
Msu was synthesized as
described by Clapp and Young (16). GSH, S-hexylglutathione,
CDNB, and 1-fluoro-2,4-dinitrobenzene (FDNB) were Sigma products.
His-tagged recombinant hGSTT2-2 and R107A mutant were expressed in
Escherichia coli and purified using immobilized metal ion
chromatography on a nickel-nitrilotriacetic acid matrix (Qiagen) as
described previously (13, 17).
Steady State Kinetic Experiments--
Kinetic experiments were
carried out at 37 °C in 1 ml of carbonate/phosphate/acetate
(50:50:50 µM) buffer (Buffer A) at suitable pH values
(between pH 5.0 and pH 10.0), containing variable amounts of GSH (from
0.2 to 100 mM) with fixed and saturating Msu concentration (0.25 mM); the reaction was started by the addition of
suitable amounts of hGSTT2-2 or of R107A mutant. The reaction rates
were measured spectrophotometrically at 298 nm and at 0.1-s intervals for a total period of 120 s. Initial rates were determined by linear regression analysis. Spontaneous reaction was negligible at all
the pH values investigated. Data of velocity versus [S] were analyzed by a rectangular hyperbole equation to yield
Vmax and Km values for GSH.
The Hill equation was evaluated for possible cooperativity. The
activity of hGSTT2-2 with CDNB or FDNB was assayed at constant
co-substrate concentration (1 mM) and variable GSH
concentration in 0.1 M potassium phosphate buffer, pH 6.5, 37 °C.
Pre-steady State Kinetic Experiments--
Rapid kinetic
experiments were performed on a Applied Photophysics Kinetic
spectrometer stopped-flow instrument equipped with a 1-cm light path
observation chamber thermostatted at 37 °C. In a typical experiment,
hGSTT2-2 or R107A mutant (30 µM), in Buffer A at a
suitable pH value, was rapidly mixed with an identical volume of GSH
(10 mM) and Msu (0.5 mM) dissolved in the same
buffer. The reaction was followed at 298 nm and performed in the 5-10 pH range. Data were fitted to Scheme I,
where E is enzyme, S is substrate, and P is product.
Acidic constants for the native enzyme were calculated by fitting
kinetic data to Equation 1,
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(Eq. 1)
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and for R107A mutant by utilizing Equation 2,
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(Eq. 2)
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Moreover, pre-steady state kinetics at different GSH
concentrations were performed by rapid mixing of the native enzyme (30 µM) dissolved in buffer A, pH 7.0, with the same volume
of buffer containing Msu (0.5 mM) and different amounts of
GSH from 0.12 to 20 mM. Data were fitted to Scheme II,
SVD Analysis of the Catalyzed Reaction of GSH with Msu--
In a
typical experiment, hGSTT2-2 (30 µM), dissolved in Buffer
A, pH 7.0, was rapidly mixed with GSH (4 mM) and Msu (0.2 mM) dissolved in the same buffer at 37 °C. 200 points
were collected on a faster time base and successively another 200 points were collected on a slower time base. Time-dependent
spectra were reconstructed from single wavelength observations (between
240 and 320 nm) by repetitively changing the wavelength following
different reagent mixing steps; a 2-nm increment step and 6-nm
bandwidth were utilized.
Optical deconvolution of time-dependent spectra sets were
performed by means of the software MATLAB (MathWorks, South Natick, MA), running on an Intel Pentium-based personal computer by using singular value decomposition (SVD) in combination with curve fitting algorithms. The matrix of time-dependent spectra (A) is
decomposed by SVD into the product of three matrices, A = UXSXVT, where the U columns are the
basis spectra, and their time dependence is represented by the V
columns. The diagonal values of the S matrix yield the relative
occupancies of the basis spectra in the data set. If a data set is
contributed by more than one optical transition, deconvolution of the
optical components (provided they have different time courses) can be
achieved by simultaneously fitting the chosen V columns subset to the
kinetic Scheme III (see below). Such best fit of the experimental data
to Scheme III, performed using either the program FACSIMILE (AEA,
Harwell, UK) or the program GEPASI 3.21 (18-20), obtaining essentially
identical results in both cases, has been carried out by means of
numerical integration, at variable steps, of the ordinary differential
equations according to Scheme III. The resulting matrix of the time
dependences of the molar fraction of the intermediate species can be
used to reconstruct (from the experimental matrix of
time-dependent spectra) the spectra of the intermediate
species at different times after mixing (21-23).
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RESULTS AND DISCUSSION |
Steady State Kinetics of hGSTT2-2 with Msu as
Co-substrate--
Steady state kinetics of hGSTT2-2 has been analyzed
at fixed and saturating Msu concentrations (0.25 mM) and
variable GSH concentrations between 0.2 and 100 mM.
Experiments were performed at 37 °C and at different pH values
between pH 5.0 and 10.0. The dependence of velocity on GSH
concentration does not obey the Michaelis-Menten equation, and hGSTT2-2
displays apparent negative cooperativity toward GSH as though the
binding of GSH to the first subunit induces a lowering of affinity in
the empty subunit. Hill plots, obtained from experiments at various pH
values, yield a Hill coefficient ranging from 0.7 to 0.8. This
non-Michaelian kinetic behavior is unexpected as the isothermal binding
of GSH has been found to be hyperbolic (10). Thus, this may correspond to a true cooperativity only if the presence of the co-substrate in the
active site causes an intersubunit communication that is absent without
it. Due to the very high affinity for Msu (Km = 5 µM), it is unclear if hGSTT2-2 displays a cooperative
behavior toward this co-substrate. For the sake of simplicity, kinetic data, at variable GSH concentration between 0.2 and 4 mM,
were only considered. They can be fit satisfactory to a simple
hyperbolic equation that gives a rough estimate of the apparent
Km(GSH)
0.5 mM. This
value does not change between pH 5.0 and pH 9.0, showing an increase
only at higher pH values. kcat value, calculated at 100 mM GSH, is low (0.15 s
1 at
pH 7.0) and scarcely pH-dependent between pH 5.0 and pH 8.0 (Fig. 1A). Under more alkaline
conditions, kcat value increases, reaching a
5-fold increase at pH 10. At this pH, the enzyme is unstable, and in a
few minutes it inactivates irreversibly. Secondary plots of
1/kcat and
Km/kcat in the presence or
absence of the enzymatic product GS-Msu (data not shown) suggest that hGSTT2-2 follows a rapid equilibrium random mechanism like the rat
isoenzyme (14).

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Fig. 1.
Effect of pH on kinetic parameters.
A, pH dependence of kcat (calculated
10 s after mixing) for hGSTT2-2 ( ) and for R107A mutant ( );
experiments were performed at 37 °C as reported under
"Experimental Procedures." The pH dependence of the pseudo-first
order kinetic constant for product formation
(k1) (B), of the first order kinetic
constant for product release (k2)
(C), and of the active enzyme concentration
(E0) (D) are obtained by stopped-flow
experiments with hGSTT2-2 ( ) and with R107A mutant ( ). The
solid lines in B are the best fit of experimental
data to Equation 1 (native enzyme) and to Equation 2 (R107A
mutant).
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As a whole, several aspects seem to characterize the
hGSTT2-2 steady state kinetics when compared with other human
GSTs as follows: (a) very low (at least 100-fold lower)
kcat and
kcat/Km(GSH) values; (b) the apparent negative cooperativity, also
observed in the Alpha and Mu GSTs, but in those enzymes it is probably the consequence of a steady state random kinetic mechanism (24); (c) kcat value is pH independent,
whereas in human Alpha, Mu, and Pi GSTs (with CDNB as co-substrate) it
parallels the deprotonation of the bound GSH (11, 25, 26).
Pre-steady State Kinetics--
Pre-steady state kinetics of
hGSTT2-2 have been dissected by means of stopped-flow
experiments performed at pH 7.0 and 37 °C. Representative time
courses of the enzymatic reaction, obtained at variable GSH
concentrations (from 0.06 to 10 mM) and in the presence of
saturating Msu (0.25 mM), are shown in Fig.
2B. The early stage of
catalysis shows a well defined burst phase of product accumulation
followed by a slower, linear phase that corresponds to the product
release. The slower portion is almost independent on GSH concentration
indicating that the product release occurs at the maximum rate even at
low GSH concentrations. The best fit of these kinetic data to the
minimal Scheme II (see "Experimental Procedures") yields apparent
microscopic rate constants reported in Table
I. It appears that
kon and koff are close to
those previously found for the formation of the binary complex
E-GSH (10). Thus, the presence of Msu does not affect the
binding of GSH, and this co-substrate interacts with the active site at
a higher or similar velocity compared with GSH.

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Fig. 2.
Time course of the GS-Msu product
formation. A, steady state kinetics of hGSTT2-2 (2 µM) performed at pH 7.0 and 37 °C in the presence of
Msu (0.25 mM) and variable GSH concentrations: 0.2 mM ( ), 1 mM ( ), 10 mM ( )
and 100 mM ( ). B, stopped-flow kinetics of
hGSTT2-2 (15 µM) performed at pH 7.0 and 37 °C in the
presence of Msu (0.25 mM) and variable GSH concentrations:
0.1 mM (a), 0.3 mM (b), 1 mM (c), and 3 mM (d).
C, stopped-flow kinetics of hGSTT2-2 at longer acquisition
times (up to 100 s); experimental conditions as in B.
The solid lines are obtained by the simultaneous fitting of
pre-steady and steady state kinetic data to Scheme III.
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Table I
Stopped-flow kinetic parameters have been calculated by fitting
pre-steady state kinetic data at variable GSH concentrations to Scheme
II
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When the linear phase following the burst is extrapolated back on the
y axis, the intercept (
) yields the effective active enzyme concentration, according to
= [E]0 (k1/(
k1+ k2))2. In
the case of hGSTT2-2, E0 corresponds to only
about 50% of the total enzyme used. This sub-stoichiometric amount of
active enzyme has been confirmed by replicate experiments with
different enzyme batches, and it seems to be a peculiarity of the human Theta isoenzyme. A second unusual finding is that
kcat calculated above by conventional
spectrophotometry (0.15 s
1), if normalized to
the effective active enzyme concentration, is 3-fold lower than
k2 (1 s
1). By using
longer acquisition times in the stopped flow apparatus (a few seconds
after mixing), the rate of product release decreases, so that
k2 approaches kcat. In
other words, the experimental traces given by the stopped-flow
apparatus are now comparable to those observed in the conventional
spectrophotometer. Thus, after the burst phase that is related to
k1, two subsequent phases characterize the
steady state kinetic behavior of T2-2. The first one occurs within
1-1.5 s after mixing (Fig. 2B); the slower second phase is
visible at longer acquisition times (see Fig. 2, A and
C).
pH Dependence of Kinetic Parameters--
The pH dependence of the
pre-steady state kinetics has been studied between pH 5.0 and 10.0 at
fixed GSH (5 mM) and Msu (0.25 mM)
concentrations. Data were analyzed according to Scheme I (see "Experimental Procedures"). Fig. 1 shows the pH dependence of the
pseudo-first order rate constant for product formation
(k1) and for the first order rate constant for
product release (k2) and the pH dependence of
the enzyme concentration (E0) calculated from
the
amplitude. Whereas E0 corresponds to
about 50% of the total protein between pH 5.0 and pH 9.0 (Fig.
1D), the pH dependence of k1 shows a
large bell-shaped trend (Fig. 1B). The best fit of the
experimental data to Equation 1 yields an apparent
pKa1 = 5.6 ± 0.20 and
pKa2 = 9.2 ± 0.20. pKa1 is close to the apparent
pKa of the bound GSH (pKa = 6.1),
as calculated in the binary complex (10). The presence of Msu in the
active site probably causes a more favorable deprotonation of the bound
GSH. The decrease of k1 above pH 8.0 suggests
that at least one protonated residue, with an apparent
pKa of 9.2, is involved in optimizing the
orientation of the substrate(s) for the chemical step.
k2, which has been calculated within 1 s
after mixing, follows a trend similar to that of
kcat, i.e. is pH independent up to pH
8.0, showing a remarkable increase only above pH 9.0 (Fig.
1C). The crystal structure of the Msu complex provides an
explanation as to why the product release is rate-limiting; the
menaphthyl ring is firmly wedged in the H-site with the C-terminal
extension completely blocking access to the surrounding solvent. The
Msu complex crystal structure was examined with a view to understanding
why product release is greatly accelerated above pH 9. No candidate
residues were identified that might be involved in loosening the
contacts of the C-terminal extension from the rest of the protein.
However, a number of residues were identified in the narrow tunnel that leads from the active site. These residues include His-40, Lys-41, Lys-53, Glu-66, Asp-104, and Cys-105. In addition to these residues, the
-amino group of GSH is another candidate. In each case the residue is in a microenvironment that might sufficiently disturb its
pKa to cause the pH-dependent kinetic
effect. In summary, the kinetic data support the previous hypothesis
that the C-terminal extension does not move to allow the passage of substrates and products, but instead a small tunnel acts as the gateway
(8).
Effect of Arg-107 Mutation on Catalysis--
In the structure of
the Msu complex, Arg-107 interacts with both GSH and the cleaved
sulfate ion (8). In the accompanying paper (10), Arg-107 has been shown
to play a crucial role in the activation of GSH since mutating this
residue causes the pKa of the thiolate anion to
shift from 6.1 to 7.8. Steady state kinetics is also modified. For
example, between pH 5.0 and pH 8.0, kcat shows a
pH dependence that contrasts with the pH invariance of the native
enzyme (Fig. 1A). Stopped-flow experiments demonstrate a
well defined burst phase only above pH 7.0, although it is completely absent under more acidic conditions. This means that below pH 7.0, the
chemical event becomes too slow, and the product does not accumulate.
The pH dependence of k1 displays an apparent
pKa of 8.1 (Fig. 1B), a value close to
the pKa of GSH in the binary complex with this
mutant (10). Thus, the replacement of Arg-107 yields a similar shift of
pKa both in the binary and in the ternary complexes
of hGSTT2-2.
It is likely that the protonated form of Arg-107 plays a role in the
chemical step by promoting not only the GSH ionization but also a
proper orientation of the substrate. In fact, in the native enzyme,
when GSH and Arg-107 are in the ionized form (between pH 7.0 and 8.0),
k1 reaches the limiting value of about 45 s
1. Conversely, the limiting value of
k1 in R107A under alkaline conditions is only 10 s
1. This value is close to that obtained in
the native enzyme at pH 10 when Arg-107 is almost uncharged, and the
bound GSH is fully deprotonated.
The slight increase of kcat and
k2 in the mutant under alkaline conditions also
suggests that replacement of Arg-107 may facilitate the product release
(Fig. 1, A and C). It was previously reported that mutation of Arg-107 had a detrimental effect on the sulfatase reaction measured in Tris/HCl, pH 8.3 (17). Conversely, under alkaline
conditions, we found a comparable or even higher activity of the Y107A
mutant compared with the wild type enzyme. This may be probably due to
the presence, in the buffers utilized, of phosphate which appears to
stabilize the Y107A mutant in a more active conformation.
A Proposed Mechanism for hGSTT2-2--
A minimal kinetic mechanism
that accounts for all pre-steady state and steady state kinetic data is
reported in Scheme III. This involves two enzyme populations
E and E' (slowly or not interconverting and each
representing about 50% of the total population) both able to bind and
activate GSH but just one competent for a fast catalytic event. The
product formation occurs in E at a rate about 100 times
faster than it occurs in E'. Product is slowly released from
both E and E', whereas it enters at a high rate
resulting in a relevant enzyme inhibition. Table
II shows the microscopic rate constants
calculated by a global fit of all pre-steady state and steady state
kinetic data and GSH binding data to the proposed mechanism. The best
fit of the experimental data to Scheme III superimpose well the
experimental traces (see Fig. 2) in the large temporal window of
0.001-100 s. The proposed kinetic mechanism accounts for the apparent
half-site behavior found in the pre-steady state analysis and also for
the apparent cooperativity observed under steady state conditions. This
mechanism is compatible either with the existence of two slowly
interconverting enzyme populations or with a single dimer population
having one subunit more active than the other. Actually, there are
extensive contacts in the dimer interface, particularly at the base of
the molecule where there are numerous hydrophobic interactions. In
comparison to other GSTs the interface is much more extensive and
includes the helical towers of the C-terminal domain and the C-terminal
tails. These latter elements also form walls of the H-sites (8). It is
thus quite conceivable that communication could exist between the
active sites, and this could be the base for a true half-site catalysis
as a result of a strong negative cooperativity.
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Table II
Stopped-flow kinetic parameters have been calculated by fitting
pre-steady state, steady state kinetic data, and binding data (10) to
Scheme III
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SVD Analysis of the Catalyzed Reaction of GSH with Msu--
The
proposed mechanism has been supported by examining the time dependence
of intermediate species (E, E', ES,
E'S, EP, E'P, and P) along the
catalytic pathway. This has been obtained by SVD analysis and best fit
of the experimental data to Scheme III as described under the
"Experimental Procedures." Fig.
3A depicts the temporal
evolution of the difference spectra (in the range 240-320 nm)
obtained after rapid mixing hGSTT2-2 with GSH and Msu. These
data, subjected to SVD analysis, were best fit to the proposed Scheme
III, employing the microscopic rate constants listed in Table II. The
time dependences of the molar fraction of the intermediate species are
reported in Fig. 3B. It is also possible to reconstruct the
spectrum of each intermediate species at a given time. Fig.
3C shows representative spectral sets at 0.001, 0.01, 0.1, and 1 s. It can be noted that both ES and
E'S are characterized by a peak below 250 nm (the intrinsic
instrumental limit did not allow us to follow accurately any optical
transition below 250 nm), which may be due to the GSH thiolate. On the
contrary, the EP and E'P species show a double
peak with a maximum at about 300 nm, which might correspond to the
product (P) as it overlaps the spectrum of synthetic GS-Msu adduct
(data not shown).

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Fig. 3.
SVD analysis of the catalyzed reaction
between GSH and Msu. A, the temporal evolution (from
0.001 to 1 s) of the difference spectrum of the reaction mixture
is shown. The spectrum was obtained by subtracting the first spectrum,
recorded 1 ms after mixing (less than 2-3% of the total burst
reaction). B shows the time courses of the species
ES, E'S, EP, E'P, and P
obtained as reported under "Experimental Procedures," according to
the minimal reaction Scheme III. Differential spectra of the optical
species were then reconstructed at different times after mixing and are
reported in C.
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It is useful now to look at the relative amount of the different
species and their spectral contribution during the approach to the
steady state phase. ES and E'S formation is
synchronous (in the millisecond time scale) to the GSH-binding event.
However, ES reaches a maximum (about 20% of the total) 10 ms after mixing, and it is absent at 100 ms when EP is the
prevalent species. E'S reaches a maximum (about 70% of the
total) after 30 ms, and it is still present (about 20%) at 1 s
after mixing. EP appears early, reaches a maximum after 100 ms and then remains populated at about 95% during the steady state
phase. E'P is slowly formed only after 100 ms and reaches a
maximum after 1 s. The product release (P) starts only after
50-100 ms and increases during the steady state phase.
As a whole, this analysis represents a time-dependent
spectral visualization of the proposed mechanism and adds new
elements to the traditional kinetic analysis. The good overlap of
the deconvoluted spectra with those of the separate species is an
intrinsic control of the correct fit of kinetic data to Scheme III.
CDNB as Substrate--
Despite previous reports (13) in our
experimental conditions, we found that hGSTT2-2 has a small but
detectable activity with CDNB, one of the best co-substrates for Alpha,
Pi, and Mu GSTs. The presence of phosphate seems to be crucial for this
activity, and at pH 6.5 and 37 °C, hGSTT2-2 displays a
kcat value of 0.07 s
1
and a Km(GSH) of about 0.6 mM. Thus its catalytic efficiency, kcat/Km, may be estimated to
be 100 s
1
M
1, about 1000 times lower than
values obtained for the recently evolved GSTs. Pre-steady state kinetic
experiments did not reveal any product accumulation before the steady
state attainment, so the rate-limiting step in catalysis would be the
chemical event or a preceding conformational transition of the ternary
complex (27). By replacing CDNB with its fluoro-analogue, FDNB,
kcat increases 40 times. Since spontaneous
reaction between FDNB and GSH is 38 times faster than that with CDNB,
the chemical step is a reasonable candidate as rate-limiting in
catalysis. It is well known that this nucleophilic aromatic
substitution occurs via an addition-elimination sequence that involves
a short-lived
-complex (27). As fluoride is a poorer leaving group
than chloride but enhances the electrophilic propensity of the C-1
atom, the
-complex formation seems to be the rate-contributing event
in hGSTT2-2 catalysis with CDNB. The remarkable increase of
kcat with FDNB also indicates that the energy
barrier due to a physical event in the catalytic pathway (product
release or structural transition) must be much lower than that of the
chemical step.
Concluding Remarks--
This paper presents a series of
investigations to dissect the hGSTT2-2 catalytic mechanism, which is
complicated by some unusual kinetic properties of this enzyme. A
multidisciplinary approach has been found essential to reach a
reasonable kinetic model; all steady state and pre-steady state kinetic
data reported here are fit well to the kinetic mechanism of Scheme III.
Even if more complicated scenarios are also plausible, this mechanism
accounts for the apparent negative cooperativity, the apparent
half-site behavior of hGSTT2-2, and the peculiar kinetic trend observed in the early stage of catalysis. Arg-107 is crucially involved in GSH
activation and in a proper orientation of GSH for catalysis. The
protonation of the guanidine side chain is an essential requirement for
its contributing role.
In addition, the SVD analysis supports the proposed mechanism giving a
reasonable spectral visualization of the species occurring along the
kinetic pathway.
Apart from the obvious interest in elucidating an unusual kinetic
mechanism, our data also present an opportunity to compare an enzyme
from an "old" GST family like hGSTT2-2 to the "younger" Alpha,
Pi, and Mu class GSTs. One crucial difference resides in the structural
rigidity of the hGSTT2-2 that may be the origin of most of the kinetic
oddities described above. A comparison of the mobility profile of
representative GST isoenzymes has been presented in the accompanying
paper (10). This comparison demonstrates the relative rigidity of the
primordial enzyme compared with the more recently evolved GSTs and, in
particular, the absence of a sequence of "cold" and "hot"
regions along the polypeptide chain. Recently, molecular dynamics
simulation data on GSTP1-1 (Pi class) revealed that segmental motions
of the protein may play a crucial role in catalysis. GSTP1-1 behaves as
a "dancing" enzyme with a breathing involving the helix
2 region
and other protein segments that produce a fast alternate opening and
closing of the active site (28, 29). A dynamic fluorescence study also
indicated GSTP1-1 exists as two families of rapidly interconverting
conformers with different binding properties (30). The interconversion is, however, very fast (with times slower than nanoseconds but much
faster than milliseconds), so that any half-site behavior or
sub-stoichiometric working active sites has not been observed. Similarly, in the human A1-1 GST (Alpha class), a rapid transition has
been proposed involving motions of the flexible C-terminal segment that
partially covers the G-site (31). Even the Mu class GST, where the
active site is confined by two flexible loops, may display a similar
fluctuating accessibility (32). On the whole, it appears that the very
similar flexibility profiles found in Alpha, Pi, and Mu GSTs (10) are
not incidental but reflect a precise evolution strategy to enhance the
catalytic efficiency of the GST superfamily. How did GSTs evolve toward
a more flexible structure? The partial or total deletion of a
cumbersome segment like the C-terminal extension of hGSTT2-2, is one
possibility, even though it causes a loss of the sulfatase activity.
Replacement of crucial residues involved in electrostatic interactions
may be a second possibility. For example, Arg-107 is likely involved in
an electrostatic interaction with Asp-104, and its replacement by Ala
induces an increased flexibility of the active site causing an
improvement of the GSH-binding properties (10). This mutation also
favors the release of product as evidenced by the slightly higher
values of k2 of the mutant (see Fig. 1). However
Arg-107 is crucially involved in the proper orientation and activation of GSH, so it was strictly conserved during the natural intra-class evolution of this enzyme.
What we found for the hGSTT2-2 catalysis using a small co-substrate
like CDNB also indicates that other evolutionary targets may have been
pursued. In fact the chemical event is largely rate-limiting in the
CDNB case; the active site is rather large and a small substrate like
CDNB could not be optimally orientated in such a site. The optimization
in catalysis could be achieved by a refinement of the substrate
orientation in the active site.