A promiscuous glutathione transferase transformed into a selective thiolester hydrolase

Article abstract of DOI:10.1039/b510115h

Human glutathione transferase A1-1 (hGST A1-1) can be reengineered by rational design into a catalyst for thiolester hydrolysis with a catalytic proficiency of 1.4 × 10⁷ M^-1. The thiolester hydrolase, A216H that was obtained by the introduction of a single histidine residue at position 216 catalyzed the hydrolysis of a substrate termed GSB, a thiolester of glutathione and benzoic acid. Here we investigate the substrate requirements of this designed enzyme by screening a thiolester library. We found that only two thiolesters out of 18 were substrates for A216H. The A216H-catalyzed hydrolysis of GS-2 (thiolester of glutathione and naphthalenecarboxylic acid) exhibits a k_cat of 0.0032 min ^-1 and a K_M of 41 M. The previously reported catalysis of GSB has a k_cat of 0.00078 min^-1 and K_M of 5 M. The k_cat for A216H-catalyzed hydrolysis of GS-2 is thus 4.1 times higher than for GSB. The catalytic proficiency (k_cat/K _M)/k_uncat for GS-2 is 3 × 10⁶ M ^-1. The promiscuous feature of the wt protein towards a range of different substrates has not been conserved in A216H but we have obtained a selective enzyme with high demands on the substrate. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b510115h

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www.rsc.org/obc | Organic & Biomolecular Chemistry
A promiscuous glutathione transferase transformed into a selective
thiolester hydrolase†
Sofia Hederos,a Lotta Tegler,b Jonas Carlsson,c Bengt Persson,c Johan Viljanena and Kerstin S. Broo*a
Received 19th July 2005, Accepted 10th November 2005
First published as an Advance Article on the web 1st December 2005
DOI: 10.1039/b510115h
Human glutathione transferase A1-1 (hGST A1-1) can be reengineered by rational design into a
catalyst for thiolester hydrolysis with a catalytic proficiency of 1.4 × 107 M−1. The thiolester hydrolase,
A216H that was obtained by the introduction of a single histidine residue at position 216 catalyzed the
hydrolysis of a substrate termed GSB, a thiolester of glutathione and benzoic acid. Here we investigate
the substrate requirements of this designed enzyme by screening a thiolester library. We found that only
two thiolesters out of 18 were substrates for A216H. The A216H-catalyzed hydrolysis of GS-2
(thiolester of glutathione and naphthalenecarboxylic acid) exhibits a kcat of 0.0032 min−1 and a KM of
41 lM. The previously reported catalysis of GSB has a kcat of 0.00078 min−1 and KM of 5 lM. The kcat
for A216H-catalyzed hydrolysis of GS-2 is thus 4.1 times higher than for GSB. The catalytic proficiency
(kcat/KM)/kuncat for GS-2 is 3 × 106 M−1. The promiscuous feature of the wt protein towards a range of
different substrates has not been conserved in A216H but we have obtained a selective enzyme with
high demands on the substrate.
Introduction
The design of novel catalysts is a challenging task that, in addition
to extending our knowledge of proteins, eventually may lead to the
development of selective and environmentally friendly catalysts1
for reactions not catalyzed by Nature. Progress in this field has
been made by rational design,2–5 computational design6–8 and
combinatorial approaches.9–14 Other studies have been performed
with enzyme models5 and through covalent modification of natural
proteins to introduce artificial functional groups.15–17
A prerequisite in the redesign of proteins is the use of an
appropriate scaffold and the glutathione transferases (GSTs
EC 2.5.1.18)ठare good candidates because of their stability,
ease of purification18 and the wealth of knowledge concerning
Scheme 1
structure–activity relationships.19 The GSTs belong to a large
family of detoxication enzymes that catalyze the conjugation of
the tripeptide glutathione (GSH) (Scheme 1) to a broad range
of different hydrophobic electrophiles.20–23 The proteins exist as
homo- or heterodimers22,24 and the active site consists of a G-
site, where GSH binds, and a promiscuous hydrophobic substrate-
binding site, the H-site, where the electrophilic molecules bind.25,26
Catalysis is mediated through a conserved tyrosine, serine, or
cysteine residue that activates GSH for nucleophilic attack22 and
the G-site is essentially conserved throughout the classes.20,26 The
differences that provide the substrate specificities of the isozymes
are mainly located in the H-site.25 In the alpha class, the H-site is
shielded from the solvent by a flexible helical segment.26
aIFM Chemistry, Division of Organic Chemistry, Linko¨ping University, SE-
581 83, Linko¨ping, Sweden. E-mail: kerbr@ifm.liu.se; Fax: +46 13 122587;
Tel: +46 13 286679
bDivision of Organic Chemistry, Biomedical Centre, Uppsala University, Box
599, SE-751 24, Uppsala, Sweden
cIFM Theory and Modelling, Division of Bioinformatics, Linko¨ping Univer-
sity, SE-581 83, Linko¨ping, Sweden
We have previously, through rational design, constructed a
thiolester hydrolase by introducing a single histidine residue in
position 216 in hGST A1-1 (Fig. 1).27 The reaction catalyzed
by the designed enzyme, A216H, is an example of catalytic
promiscuity28–30 where an alternative function is revealed by a
minimal change of active site residues. The A216H-catalyzed
hydrolysis of the synthetic substrate GSB (Scheme 1) to form GSH
and benzoic acid showed a catalytic proficiency31 (kcat/KM)/kuncat
of more than 107 M−1.27 The wt protein is instead covalently
modified with the acyl moiety on residue Y9.32 This acylated Y9 is
† Electronic supplementary information (ESI) available: Stability of
A216H as measured by CDNB activity; the acetonitrile gradients used
in the HPLC analyses; diagrams and time points from saturation kinetics
HPLC measurements. See DOI: 10.1039/b510115h
‡ Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene; GSH, glutathione,
c-Glu-Cys-Gly; GST, glutathione transferase; hGST A1-1, GST A1-
1 isoform from human; G-site, glutathione-binding site; GS-thiolester,
thiolester of glutathione; HPLC, high performance liquid chromatogra-
phy; H-site, hydrophobic electrophile binding site; MALDI-MS, matrix
assisted laser desorption mass spectrometry; NaPi, sodium phosphate;
TFA, trifluoroacetic acid; UV, ultraviolet; wt, wild-type.
§ This nomenclature is derived from that recommended by Mannervik.45
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Fig. 1 A close-up of the crystal structure (1GUH) of hGST A1-1 in a complex with S-benzyl-glutathione.26 The amino acids Y9 and A216 are shown
in “stick” representation and helix 9 (a9) is also indicated. The protein is dimeric but shown here as a monomer for reasons of clarity of presentation.
an intermediate on the reaction pathway in the A216H-catalyzed
hydrolysis reaction of GSB.27
This experiment also provided the retention time of each acid. To
maximize the yields of the reactions, the samples were incubated
for an extended period of time (26 h), something that is possible
due to the stability of A216H as measured by the CDNB activity
that is constant over at least 72 hours incubation at 25 ◦C
(ESI†). As discussed in our previous study, the uncatalyzed
hydrolysis reactions of the substrates were almost negligible.27,33
All compounds in the library were incubated separately in all
experiments either with or without protein. The reaction mixtures
were quenched with trifluoroacetic acid and analyzed by reversed
phase HPLC following addition of internal standard.
Since the natural detoxication activity of wt hGST A1-1 includes
a wide range of substrates,25 we were interested in investigating the
substrate requirements of the novel thiolester hydrolase A216H. A
panel of 17 thiolesters of glutathione (GS-thiolesters) was used
as a library in screening experiments. The library contains GS-
thiolesters that vary in size, shape, reactivity and hydrophobicity.33
Two non-aromatic GS-thiolesters were included since GST A1-1
from rat (rGST A1-1), a protein highly homologous to hGST A1-
1, has been shown to hydrolyze non-aromatic thiolesters.34 This
library has previously been used to investigate the requirements for
the site-specific Y9 modification of wt hGST A1-1.33 In that study
78% of the GS-thiolesters were able to site-specifically modify Y9
and since the same library was used in this study, we hoped to
explore the requirements for catalysis versus modification.
Selection criteria
The selection criteria of the screening procedure were initially
generous and a stepwise sharpening of the analytical processing
filtered out false positives. This procedure also automatically in-
cluded repeated measurements of potential substrates even though
a screening process was used. We had previously determined27
that 5.6 lM benzoic acid was produced after 24 h using 5 lM
A216H and 75 lM GSB. With the detection limits taken into
account, a higher concentration (15 lM) of A216H was used in
screening experiment A to allow the identification of substrates
that displayed at least 25% of the rate constant of A216H-catalyzed
hydrolysis of GSB. The selection criteria in screening experiment
A were generous and samples that showed any trace of acid with
a signal-to-noise ratio larger than 10 were further analyzed in
screening experiment B.
In screening experiment B the concentrations of A216H and
GS-thiolester were lowered to match previous studies performed
with A216H27 since we intended to analyze the reactions with UV
spectroscopy. The lowered concentration of A216H from 15 lM
to 5 lM placed higher demands on the substrate. To elucidate the
importance of the histidine residue in position 216, samples of GS-
thiolesters incubated with wt protein were included in screening
experiments B–D. Background samples of the GS-thiolesters
incubated without protein were also examined. A threshold was
set in experiment B so that there had to be a difference of 65%
or more in relative concentration for A216H compared to the wt
sample in either GS-thiolester consumption or acid production.
This threshold was set to allow for the fact that the wt protein
Results
To analyze all GS-thiolesters (18 including GSB) with five time
points per sample, would generate more than 300 injections when
using reversed phase HPLC (samples incubated with protein
and reference samples). This is a time-consuming process (ap-
proximately 15 000 min of HPLC time) and, in addition, the
measurements would have been performed only once. We have
instead used an alternative method with a screening approach
where the selection criteria have been sharpened in every new step.
In total, four screening experiments (A–D) were performed to
verify the results and to elucidate the substrate requirements of
the designed enzyme A216H.
The reaction products of the hydrolysis reaction would be
GSH and the corresponding acid (Scheme 1) but we have only
tried to detect either the decrease of substrate (GS-thiolester) or
formation of acid. This is because GSH was very difficult to detect
quantitatively in our pilot experiments using reversed phase HPLC
due to its high solubility in water and thus poor and unreliable
retention times.35 In addition, the thiol is prone to oxidation and
this also makes quantitative HPLC analyses difficult.
Prior to starting the screening experiment, we determined that
a common detection limit for the acids was approximately a
few lM using our HPLC method with detection at 250 nm.
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reacts covalently in a stoichiometric fashion with 13 of the 17 GS-
thiolesters in the panel.33 In addition, it has previously been shown
that the wt protein hydrolyzes some non-aromatic GS-thiolesters36
and the threshold was also set to filter out such GS-thiolesters.
The stringency in experiment C was increased by the use of
a kinetic analysis of the samples. The observed rate constants
for both GS-thiolester consumption and acid production had to
display a difference of 65% or more for the A216H-incubated
sample compared to the sample incubated with wt protein.
The final screening experiment, D, was mainly performed to
resolve the ambiguity concerning one of the GS-thiolesters (GS-3).
Table 1 Summary of screening experiments A–D. ✗ denotes that the GS-
thiolester has participated in the screening experiment
Screening experiment
GS-thiolester
A
B
C
D
Saturation kinetics
a
GSB
✗
✗
✗
✗
✗
✗
✗
✗
✗
✗
✗
✗
✗
✗
✗
✗
✗
✗
✗
GS-1
GS-2
GS-3
GS-4
GS-5
GS-6
GS-7
GS-8
GS-9
GS-10
GS-11
GS-12
GS-13
GS-14
GS-15
GS-16
GS-17
✗
✗
✗
✗
✗
✗
✗
✗
✗
✗
✗
Results from the screening experiments
✗
✗
✗
✗
A summary of the outcome of the screening experiments is
presented in Table 1. In screening experiment A, nine of the GS-
thiolesters (Table 1) and the control substrate GSB displayed traces
of acid in the presence of A216H (Figs. 2A and B). The previously
thoroughly investigated substrate GSB27 was included as a
control (Fig. 2A) to verify that the reaction conditions used were
satisfactory and that the protein was in good shape. The analyses
were done at various wavelengths based on the UV spectrum
✗
✗
a From ref. 27.
Fig. 2 Representative data from the screening experiments A–D. In screening experiment A, 15 lM A216H was incubated with 100 lM GS-thiolester,
and analyzed by reversed phase HPLC. Traces are shown from (A) the positive control GSB (k = 229 nm) and (B) GS-2 (k = 250 nm). The GS-thiolester
and the acid reference are shown as well. The observed peak from the product (A) benzoic acid and (B) naphthalenecarboxylic acid (2) is indicated
with an arrow. (C) Data from GS-2 obtained in screening experiment B. The thiolester GS-2 (75 lM) was incubated with 5 lM A216H and wt protein
respectively. A background sample with no protein present was also collected. Late time points (after 46 hours of incubation) were analyzed by reversed
phase HPLC and the peak areas were derived from integration of the peaks in the chromatograms. The increased production of naphthalenecarboxylic
acid in the A216H sample compared to the wt and reference samples is seen. (D) In screening experiment C, a kinetic analysis was performed with both
early and late time points that were analyzed by reversed phase HPLC. Here, the peak area of remaining GS-2 divided by the peak area of the internal
standard is plotted as a function of time. (E) Data from screening experiment D that was derived from HPLC analysis of 5 lM A216H incubated with
75 lM GS-2. The concentration of GS-2 was obtained by using relative responses and is plotted as a function of time.
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of each acid and GS-thiolester (typically 229 and 250 nm)
since the HPLC was equipped with a photo diode detector.
However, a peak from the protein was seen in close proximity to
the internal standard at 229 nm (Fig. 2A) so analysis was made
at 250 nm when allowed by the UV spectrum of each compound.
The remaining eight GS-thiolesters that did not show any trace of
acid were excluded from further experiments.
The reactions in screening experiment B were followed by
UV spectroscopy but due to the complexity of the resulting
UV traces no effort was put into the interpretation of the
data. Instead, late time points were collected after approximately
two days and analyzed by reversed phase HPLC. The peaks in
the chromatograms were integrated and the relative concentration
was determined by dividing the area of the GS-thiolester or acid
peak respectively by the area of the internal standard (Fig. 2C).
Five of the GS-thiolesters (Table 1) fulfilled the selection criteria
and were further analyzed in experiment C. The remaining four
GS-thiolesters were rejected.
The kinetic analysis in screening experiment C is illustrated
in Fig. 2D. Only one GS-thiolester (GS-2) fulfilled the selection
criteria. The data from GS-3 were irreproducible but GS-3 showed
interesting results and was further analyzed by conducting a fourth
screening experiment (D).
Fig. 3 A216H-catalyzed hydrolysis of GS-2 to form GSH and naph-
thalenecarboxylic acid. The kinetic studies were performed with 5 lM
A216H at pH 7 and 25 ◦C and the reaction rates were determined by
analyzing five time points by reversed phase HPLC. The peaks of GS-2
and the internal standard (IS) were integrated to determine the remanining
concentration of GS-2. HPLC traces (inset) show the decrease of GS-2
and formation of naphthalenecarboxylic acid (2) in the prescence of
A216H. The decrease of GS-2 in the wt sample is due to acylation of
Y9.33
The final screening experiment (D) was carried out with GS-2
and GS-3. The concentration of GS-3 was varied in an attempt
to resolve the ambiguities concerning the irreproducible results.
For all samples, five time points were withdrawn and analyzed
by reversed phase HPLC. The concentration of remaining GS-
thiolester was calculated, using relative responses, and plotted
as a function of time (Fig. 2E) to determine the rate constants.
Due to the low signal-to-noise ratio of the acids 2 and 3, only
the decrease in GS-thiolester concentration was used. Again,
experiments performed with GS-3 were not reproducible and GS-
3 was therefore excluded as a possible substrate. Hence, only GS-2
was found to be a substrate for A216H.
reagent and hGST A1-1 is thus not a catalyst for hydrolysis of
GS-2 as shown by the inset in Fig. 3.
The background hydrolysis of GS-2 was determined using N-
acetylated GS-2. This is due to a previous observation with GSB
where a competing intramolecular acyl transfer reaction to form
N-acylated GSH proceeds with a rate of 5.8 × 10−9 M min−1
when no protein is present. The first-order rate constant of the
uncatalyzed hydrolysis of N-acetylated GS-2 was determined by
reversed phase HPLC to be 3 × 10−5 min−1 at pH 7 (100 mM
NaPi) and 25 ◦C. The rate constant for hydrolysis of N-acetylated
GSB has previously been determined to be of the same order of
magnitude (1.1 × 10−5 min−1).27
Saturation kinetics of A216H
The Michaelis–Menten behavior of A216H towards GS-2 was
analyzed by incubating various concentrations of GS-2 with 5 lM
A216H at pH 7 and 25 ◦C. The hydrolytic reaction to form
naphthalenecarboxylic acid (2) and GSH from GS-2 was followed
by monitoring the consumption of GS-2 by reversed phase HPLC
at five time points (ESI†). The reaction followed saturation kinetics
under turnover conditions and the kinetic profile is seen in Fig. 3.
The kinetic parameters were determined to be kcat = 0.0032 min−1
and KM = 41 lM (Table 2). The wt protein is acylated at Y9 by
GS-233 but the reaction stops after addition of one equivalent of
Computer simulation studies
The distance between the epsilon nitrogen of H216 in A216H and
the carbonyl carbon of the substrate was investigated by molecular
modeling to obtain supporting information about why catalysis
occurs for only two out of 18 GS-thiolesters tested. The acyl groups
of nine representative GS-thiolesters (Table 3) were attached
covalently to the side chain of Y9 using the crystal structure of
A216H27 (Fig. 4) as a starting point. Energy minimizations of
the modified structures were then done by Monte Carlo methods
and the observed distances between the epsilon nitrogen (Ne2) of
H216 and the carbonyl carbon (Ch) of the Y9 ester are shown in
Table 3. The only GS-thiolesters in this subset that show distances
Table 2 A216H-catalyzed hydrolysis of GSB and GS-2
Parameter
cat/min−1
GSBa
GS-2
˚
less than 5 A between Ne2 and Ch are GSB and GS-2. It should be
k
0.00078
5
0.0032
41
K
M/lM
noted that acyl groups that do not display reoccurring structures
are probably not in the lowest energy conformation since several
different structures were found when repeating the simulations.
Out of the nine acids modeled at Y9, five show reoccurring
structures, where typically half the simulations have very similar
conformations and energies.
k
uncat/min−1
1.1 × 10−5
3 × 10−5
kcatKM−1/M−1 min−1
156
78
−1
kcatkuncat
71
107
(kcatKM−1)/kuncat−1/M−1
1.4 × 107
2.7 × 106
a From ref. 27.
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Fig. 4 Close-up of the active site of the crystal structure (1USB) of A216H27 with (A) benzoic acid and (B) naphthalenecarboxylic acid introduced by
˚
˚
computer modeling at the side chain of Y9. The distance between H216 and the carbonyl carbon of the Y9 ester is (A) 4.6 A and (B) 4.0 A.
Table 3 Distances between the Ne2 atom of H216 and the Ch atom of
acylated Y9 in A216H
each round. The reasons behind the false positives are complex
but the worst problems are probably low S/N ratios of the acid
products, and acylation of Y9 without subsequent hydrolysis.
Using this screening procedure we were able to analyze all the
potential substrates in the library with a mere 160 injections
instead of >300 as outlined in the Results section.
Recently, the same GS-thiolesters (Chart 1) were also used
to obtain information about the requirements for site-specific
modification of Y9 in wt hGST A1-1, a step that corresponds to
the first step in the catalytic cycle of A216H-catalyzed hydrolysis
of GSB. We found that 14 of the 18 GS-thiolesters (78%) in the
library (including GSB) were able to acylate Y9.33 The generality
was perhaps not so surprising given that the natural function of the
GSTs is conjugation of glutathione to a wide range of electrophiles
and the H-site is thus accordingly promiscuous.20–23 Interestingly,
we now observe that A216H is only able to hydrolyze two out
of the 18 GS-thiolesters tested (10%). Even though several of
the GS-thiolesters were able to acylate Y9 as demonstrated by
decreases in the concentration of starting material, only in the case
of GS-2 (and GSB) is this a true intermediate that allows H216
to complete the catalytic cycle. This demonstrates that correct
orientation of the catalytic residues is necessary during the whole
cycle. In retrospect, we were indeed fortunate when we designed
and synthesized GSB and not a similar substrate. This further
illustrates the difficulties encountered when trying to design novel
enzymes; it is not only the protein but also the substrate that needs
to be taken into account. Perhaps the best starting point would be
to investigate a whole panel of substrates and not just one or a few
to evaluate the effect of a certain mutation.
The library contains a range of different GS-thiolesters with
respect to size, shape, hydrophobicity and pKa of the acid.33
The non-aromatic GS-thiolesters (GS-15 and GS-16) were also
included in the library since the wt rat GST A1-1 (closely
homologous to hGST A1-1) is capable of hydrolysis of non-
aromatic GS-thiolesters.34 Even though the library contains this
variance, only two GS-thiolesters are substrates for A216H and
it is thus not possible to draw any relevant conclusions about
the substrate requirements. For example, the difference between
GS-1 and GS-2 is very small and the substitution in position 5
of GS-1 may perhaps possess a sterical hindrance. It would be
Estera
Distance Ne2–Ch/A
Reoccurring structureb,c
˚
Tyr-B
Tyr-2
Tyr-3
Tyr-4
Tyr-8
Tyr-10
Tyr-11
Tyr-12
Tyr-13
4.6
4.0
5.1
6.6
6.1
5.9
8.2
5.2
6.2
+
−
−
+
+
−
+
−
+
a B corresponds to benzoic acid and the numbers to the acids in Chart 1.
b
˚
Several simulations have given the same conformation (rmsd < 0.6 A)
with similar energies. c + corresponds to yes and − to no.
Discussion
We have previously reported that hGST A1-1 can be reengineered
into a thiolester hydrolase by a single point mutation, A216H.27
This new enzyme catalyzed the hydrolysis of GSB to form GSH
and benzoic acid via an Y9-acylated intermediate. The wt protein is
also acylated at Y9 by GSB but the reaction stops at that stage. The
introduced histidine thus opens up a new reaction pathway.27 In a
separate line of investigation we found that Y9 in the wt protein
could be acylated with a range of different GS-thiolesters33 and
we were thus encouraged to explore the substrate requirements of
A216H in order to investigate if the introduction of a histidine
residue could open up the hydrolytic pathway for GS-thiolesters
other than GSB.
Only two out of 18 GS-thiolesters are substrates for A216H
Four successive screening experiments filtered out GS-2 as the
only new substrate in the library. In each experiment a number of
potential substrates were eliminated but false positives survived
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Chart 1
interesting to use a more refined library based on GSB where
others. The naphthylated side chain of Y9 may, for example, bind
better in the active site than the benzoylated one and thus give rise
to a higher activation energy.
methyl groups are introduced in various positions in the aromatic
ring. A primary choice would, for example, be 3,4-dimethylbenzoic
acid that closely resembles GS-2.
The kcat(GS-2)/kcat(GSB) is 4.1
The distance between acylated Y9 and H216 is the shortest for the
two successful substrates
The kinetic parameters derived from the saturation kinetics studies
show that the ratio kcat(GS-2)/kcat(GSB) is 4.1. A higher kcat indi-
cates that the introduced histidine 216 possibly is more favorably
oriented for catalysis of hydrolysis of GS-2 than GSB. That the
KM for GS-2 should be higher (41 lM) than that for GSB (5 lM)
is somewhat surprising since naphthalenecarboxylic acid (2)
seemingly has the potential to interact more favorably with the
hydrophobic H-site of A216H due to its larger aromatic system.
However, it should be noted that KM cannot be treated as a pure
dissociation constant (KD) in a multistep reaction. Again, the
conclusion is that it is not easy to a priori model the outcome
of the dynamic process of catalysis.
The computer simulations support our experimental finding that
only GSB and GS-2 are substrates for A216H. The carbonyl
carbons of the esters formed from Y9 and benzoic acid and
naphthalenecarboxylic acid (2), respectively, are the only ones
˚
that are within a distance of 5 A with respect to the epsilon
nitrogen of H216 (Table 3). In the case of GSB a reoccurring
˚
structure appeared (Fig. 4A) with a distance of 4.6 A between the
epsilon nitrogen of H216 and the carbonyl carbon of the ester
formed from benzoic acid and Y9. In the case of GS-2 (Fig. 4B)
no reoccurring structure was obtained, however, the structure
with lowest energy (out of 14 simulations) has been used for a
qualitative comparison. The corresponding distance in the case
The background hydrolysis rates of GSB and GS-2 were
obtained using N-acetylated GS-thiolesters since we previously
found that GSB does not hydrolyze even after nine days at pH 7
˚
of GS-2 was 4.0 A. This suggests that only for GSB and GS-2 is
◦
the histidine residue in the correct orientation to aid in catalysis
and it again stresses that precise positioning of the catalytic amino
acids is of utmost importance in the design of novel enzymes.
However, other factors may also contribute to the discrimination
mechanism. For example, the positioning of the acyl intermediate
may render some of the intermediates to be more stabilized than
and 25 C but rather undergoes a rearrangement reaction where
the acyl group migrates to the a-amino group of GSH.27 Thiolester
hydrolysis is not expected to be affected by N-acetylation. The cat-
alytic proficiency (kcat/KM)/kuncat of A216H-catalyzed hydrolysis
of GS-2 was calculated to be 3 × 106 M−1. Thus, the higher kcat
compensates for the higher KM.
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The suggested mechanism for A216H-catalyzed hydrolysis of GS-2
rically using e280(hGST A1-1) = 24700 M−1 cm−1).38 A standard
assay39 using 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and
1 mM GSH in 100 mM NaPi pH 6.5 at 30 ◦C was conducted
to check the activity of the proteins.
Through detailed mechanistic studies27 we determined that an
acylated Y9 is an intermediate in the A216H-mediated hydrolysis
of GSB. It is highly likely that the mechanism of hydrolysis of GS-
2 by A216H also proceeds via an acylated Y9-intermediate. The
basis of this assumption is that the wt protein becomes modified
at Y9 by both GS-2 and GSB as shown by HPLC and MALDI-
MS.32,33 The introduced histidine residue in A216H is crucial for
the catalytic cycle to proceed and in the case of GSB it almost
certainly functions as a general base and not a nucleophile.27 We
find it highly likely that the same type of mechanism operates in
the case of GS-2, especially since this is the only new substrate
found during the screening process and it is not likely that the
amino acids are positioned such that the mechanism would switch
to nucleophilic catalysis by the histidine.
Screening procedures
The screening procedures are illustrated in Fig. 5.
Conclusion
The substrate requirements of the designed enzyme A216H have
been investigated through a combinatorial screening process of
17 GS-thiolesters. Only one new substrate, GS-2, was found and
the ratio kcat(GS-2)/kcat(GSB) is 4.1. The ratio of the catalytic
proficiencies (GS-2/GSB) is 0.2. The promiscuous feature of the wt
protein towards a range of different natural substrates has not been
conserved in the designed enzyme A216H but we have obtained a
selective enzyme with high demands on the substrate. We believe
that our findings illustrate the challenges encountered in rational
design of novel protein function and may be of importance for
future biotechnological applications using GSTs.
Experimental
All chemicals and reagents used were of the highest purity
available. The synthesis of the GS-thiolesters has previously
been reported33 and in this study the same stock solutions were
used. The HPLC experiments were performed with a Varian
system (ProStar 410 autosampler together with a ProStar 230
delivery system and a ProStar 330 photodiode array detector)
using a Kromasil C8 column (4.6 mm × 250 mm, Supelco
Inc, Bellefonte, USA) and shallow acetonitrile gradients with
aqueous trifluoroacetic acid (0.1%) (ESI†). The HPLC samples
were mixed with the internal standard 2-chloro-4-nitrophenol and
trifluoroacetic acid to a final volume of 30 lL and stored at −20 ◦C
until analysis. The chromatograms in screening experiment B–D
and in the saturation kinetic studies were integrated using the
Varian software. The UV measurements were carried out with a
Varian Cary-100 UV-visible spectrophotometer and submicrocells
(Varian, Palo Alto, USA).
Fig. 5 A flow chart of the experimental procedures used for screening
experiments A–D.
Saturation kinetics
The saturation behavior of A216H towards GS-2 was analyzed
by mixing 5 lM A216H with different concentrations of GS-2 in
100 mM NaPi pH 7 and 25 ◦C. Time points (4–6 per sample)
were collected and analyzed by reversed phase HPLC. Due to the
high concentration of enzyme relative to substrate, the Michaelis–
Menten equation in the form in which no assumption of [E] «
[S] has been made was used to analyze the results, eqn (1).40 The
kinetic parameters, kcat and KM, were determined using eqns (2)
Site-directed mutagenesis and protein expression
The A216H mutant was prepared by inverse PCR using the
wt vector pGNdeA137 and custom-synthesized primers (DNA
Technology, Aarhus, Denmark) as described previously.27 The wt
protein and the mutant A216H were expressed in Escherichia coli
BL21(DE3) (Novagen, San Diego, USA) and purified by cation-
exchange chromatography using a HiTrap SP column (Amersham
Biosciences, Piscataway, USA).38 SDS-PAGE was used to confirm
the purity and concentrations were determined spectrophotomet-
and (3).
m = kcat/2(KM + [E]0 + [S]0
√
−
[(KM + [E]0 + [S]0)2 − 4[E]0[S]0])
(1)
(2)
(3)
kcat = Vmax/[E]0
KM = Vmax/2
9 6 | Org. Biomol. Chem., 2006, 4, 90–97
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The saturation kinetics in the case of GSB was previously
determined by monitoring the disappearance of the thiolester
functionality of GSB at 266 nm by UV spectroscopy.27
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Background hydrolysis rate
N-Acetylated GS-2 was synthesized by adding four portions of
10 equivalents of sulfo-N-hydroxysuccinimide acetate during a
period of four hours to GS-2 (final concentration 75 lM) in
100 mM NaPi pH 7. HPLC was used to confirm the acetylation
of GS-2. The background hydrolysis sample was incubated at
25 ◦C and time points were collected over 11 days and mixed with
internal standard and trifluoroacetic acid to quench the reaction
whereupon the samples were stored frozen until analysis. Reversed
phase HPLC was used to determine the remaining concentration
of N-acetylated GS-2.
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The PDB file 1USB was used as a starting structure for the
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˚
sphere of 5 A around Y9. Remaining residues of the molecule were
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tion, but in order for the Monte Carlo method to work there must
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Acknowledgements
We thank Professor B. H. Jonsson (Linko¨ping University) for
critical reading of the manuscript. This work was financially
supported by The Wenner-Gren Foundation, The Carl-Trygger
Foundation, The Knut and Alice Wallenberg Foundation, and
the Swedish Research Council. S.H. is enrolled in the graduate
school Forum Scientium, supported by the Swedish Foundation
for Strategic Research (SSF).
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