Temperature and pH dependence of Enzyme-catalyzed hydrolysis of trans-methylstyrene oxide. A unifying kinetic model for observed hysteresis, Cooperativity, and regioselectivity

Article abstract of DOI:10.1021/bi902157b

The underlying enzyme kinetics behind, the regioselective promiscuity shown by epoxide hydrolases toward, certain epoxides has been studied. The effects of temperature and pH on regioselectivity were investigated by analyzing the stereochemistry of hydrolysis products of (1R,2R)-trans-2-methylstyrene oxide between 14-46 °C and pH 6.0-9.0, either catalyzed by the potato epoxide hydrolase StEH1 or in the absence of enzyme. In the enzyme-catalyzed reaction, a switch of preferred, epoxide carbon that is subjected to nucleophilic attack is observed at pH values above 8. The enzyme also displays cooperativity in substrate saturation plots when assayed at temperatures ≤30 °C and at intermediate pH. The cooperativity is lost at higher assay temperatures. Cooperativity can originate from, a kinetic mechanism involving hysteresis and will be dependent on. the relationship between K_cat and the rate of interconversion between two different Michaelis complexes. In the case of the studied reactions, the proposed different Michaelis complexes are enzyme-substrate complexes in which the epoxide substrate is bound in different binding modes, allowing for separate pathways toward product formation. The assumption of separated, but interacting, reaction pathways is supported by that formation of the two product enantiomers also displays distinct pH dependencies of K_cat/ K_M The thermodynamic parameters describing the differences in activation enthalpy and entropy suggest that (1) regioselectivity is primarily dictated by differences in activation entropy with positive values of both ΔΔ^?H and ΔΔ ^? and (2) the hysteretic behavior is linked, to an interconversion between Michaelis complexes with rates increasing with temperature. From the collected data, we propose that hysteresis, regioselectivity, and, when applicable, hysteretic cooperativity are closely linked properties, explained by the kinetic mechanism earlier introduced by our group.

Full text of DOI:10.1021/bi902157b

Biochemistry 2010, 49, 2297–2304 2297
DOI: 10.1021/bi902157b
Temperature and pH Dependence of Enzyme-Catalyzed Hydrolysis of
trans-Methylstyrene Oxide. A Unifying Kinetic Model for Observed Hysteresis,
†
Cooperativity, and Regioselectivity
‡
Diana Lindberg, Mario de la Fuente Revenga, and Mikael Widersten*
Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden.
Current affiliation: Instituto de Quimica Medica-CSIC, Juan de la Cierva 3, E-28006 Madrid, Spain.
‡
Received December 16, 2009; Revised Manuscript Received February 9, 2010
ABSTRACT: The underlying enzyme kinetics behind the regioselective promiscuity shown by epoxide
hydrolases toward certain epoxides has been studied. The effects of temperature and pH on regioselectivity
were investigated by analyzing the stereochemistry of hydrolysis products of (1R,2R)-trans-2-methylstyrene
oxide between 14-46 °C and pH 6.0-9.0, either catalyzed by the potato epoxide hydrolase StEH1 or in the
absence of enzyme. In the enzyme-catalyzed reaction, a switch of preferred epoxide carbon that is subjected to
nucleophilic attack is observed at pH values above 8. The enzyme also displays cooperativity in substrate
saturation plots when assayed at temperatures e30 °C and at intermediate pH. The cooperativity is lost at
higher assay temperatures. Cooperativity can originate from a kinetic mechanism involving hysteresis and will
be dependent on the relationship between kcat and the rate of interconversion between two different Michaelis
complexes. In the case of the studied reactions, the proposed different Michaelis complexes are enzyme-sub-
strate complexes in which the epoxide substrate is bound in different binding modes, allowing for separate
pathways toward product formation. The assumption of separated, but interacting, reaction pathways is
supported by that formation of the two product enantiomers also displays distinct pH dependencies of kcat
/
K . The thermodynamic parameters describing the differences in activation enthalpy and entropy suggest
M
that (1) regioselectivity is primarily dictated by differences in activation entropy with positive values of both
‡
‡
ΔΔH and ΔΔS and (2) the hysteretic behavior is linked to an interconversion between Michaelis complexes
with rates increasing with temperature. From the collected data, we propose that hysteresis, regioselectivity,
and, when applicable, hysteretic cooperativity are closely linked properties, explained by the kinetic
mechanism earlier introduced by our group.
Epoxides are important compounds in the synthesis of fine
chemicals and pharmaceutical intermediates (1). Epoxide hydro-
lases facilitate ring-opening reactions of the epoxide ring struc-
ture, incorporating a water to yield a vicinal diol. The soluble
epoxide hydrolase (EC 3.3.2.10) from Solanum tuberosum,
StEH1, belongs to the structural family of R/β-hydrolase fold
enzymes (2). The chemical mechanism of R/β-hydrolase fold
epoxide hydrolases involves the formation of a covalent alkyl
enzyme intermediate resulting from a nucleophilic attack by an
Asp carboxylate on one of the oxirane carbons, facilitated by
tyrosine phenols hydrogen-bonded to the epoxide oxygen. The
reaction is concluded by a base-catalyzed hydrolysis of the alkyl
enzyme intermediate into the vicinal diol product (3-6). The
extent of selectivity shown by epoxide hydrolases for different
epoxide substrate stereoisomers is dependent on (1) the enantios-
electivity and (2) the regioselectivity shown in the initial nucleo-
philic attack on the oxirane ring (Figure 1).
marily attacked at the least hindered carbon while the regios-
electivity for mono- or 1,2-disubstituted aliphatic substrates may
differ between substrate enantiomers (29). The selectivity of the
reactions with phenyl-substituted epoxides are more complicated
to predict due to the ability of the phenyl ring to stabilize a
carbocation-like reaction intermediate (15, 30, 31). StEH1 dis-
plays a relatively broad substrate specificity range but with a
preference for aliphatic and aromatic trans-substituted epoxi-
des (3, 12, 32-37). While the enzyme shows a high degree of
regioselectivity with most compounds, it displays promiscuous
behavior in the hydrolysis of certain aryl epoxides. In the present
context, promiscuity implies that the enzyme may catalyze attack
at either electrophilic carbon of the epoxide ring. A clear-cut
example is the transformation of styrene oxide, where the (S)-
enantiomer is attacked at the more stabilized benzylic carbon
whereas the (R)-isomer is almost exclusively reacting at the
sterically least hindered aliphatic carbon (37). With trans-2-
methylstyrene oxide (2-MeSO), the (1S,2S)-enantiomer (1) is
exclusively attacked at the benzylic carbon while the (1R,2R)-
enantiomer (2) is opened at either carbon (Figure 1) (12). A
similar promiscuity with the 2-MeSO epoxide is also observed in
reactions catalyzed by a cress epoxide hydrolase (AtEH) and by
other isoenzymes (7, 11, 13, 38, 39). Notably, in the AtEH-
catalyzed reaction the enantiospecificity is reversed as compared
to the StEH1 reaction; 2 is exclusively reacting at the benzylic
The regioselectivity toward a specific epoxide is influenced by
the epoxide hydrolase catalyst and by intrinsic properties of the
substrates (7-29). 1,1-Disubstituted aliphatic epoxides are pri-
†
This work was supported by The Swedish Research Council, The
Carl Trygger Foundation, and The O. E. and Edla Johansson Founda-
tion.
*Corresponding author. E-mail: mikael.widersten@biorg.uu.se. Phone:
þ46 (0)18 471 4992. Fax: þ46 (0)18 55 8431.
r 2010 American Chemical Society
Published on Web 02/10/2010
pubs.acs.org/Biochemistry

2
298 Biochemistry, Vol. 49, No. 10, 2010
Lindberg et al.
quantitative measurements of product diol ratios, have allowed
us to extract kinetic parameters for formation of the individual
product enantiomers starting from the same substrate enantio-
mer. The study also aimed at investigating possible linkages
between conformational changes in the enzyme, hysteresis, and
degree of regioselectivity.
EXPERIMENTAL PROCEDURES
Chemicals, Reagents, and General Procedures. 2-MeSO
and other chemicals were purchased from Aldrich at the highest
available purity. The StEH1 enzyme was overexpressed in
Escherichia coli XL1 Blue cells (Stratagene) and purified accord-
ing to a previously described protocol (3). Once purified, the
proteins retained full activity upon storage at 4 °C over the time
period of analysis. In the HPLC measurements, separation of
analytes was performed over a Daicel Chiralpak AD-H (250 ꢀ
F
IGURE 1: Stereoselective outcome of StEH1-catalyzed reaction of
the two enantiomers of trans-2-MeSO. The StEH1 regioselectivity
toward 1 is >98% in preference of C-1, while for enantiomer 2 the
enzyme shows regioselective promiscuity and prefers C-1 to C-2 with
a factor of 2:1 at pH 7.5, 30 °C.
Scheme 1
4
.6 mm i.d.) using a Shimadzu LC10 pump. Substrates were injected
with a Shimadzu SIL-10AF autosampler, and peaks were detected
at 220 nm using a Shimadzu SPDM20A diode array detector.
The change in the molar absorbance resulting from hydrolysis of
2-MeSO to diols was determined by subtracting the UV spectrum
of the formed diol, at equilibrium, from that of the epoxide,
obtaining a molar extinction coefficient of Δε = -4.3 mM- cm
1
-1
.
The equilibrium absorbance of the diol was achieved by hydrolyz-
ing 0.2 mM 1 to completion in the presence of 0.4 μM StEH1 in
0
.1 M sodium phosphate, pH 7.5. Buffer pH was adjusted by
epoxide carbon whereas 1 is attacked at either carbon with low
preference (13, 38). Theoretical investigations on the reaction
with 2-MeSO catalyzed by the murine soluble epoxide hydrolase
suggest that regioselectivity is dependent on a hydrogen bond
between one of the active site tyrosines with the substrate epoxide
oxygen (13, 40). The results from the molecular dynamics
simulations by Schøitt and Bruice proposed that the rigidness
of the active site is important in determining the degree of
regioselectivity, with a tight active site conformation favoring a
higher degree of regioselectivity (40).
adding either NaOH or H PO to the sodium phosphate solution.
3
4
All curve fitting and data statistic analyses were performed with
programs MMFIT, INRATE, RFFIT, LINFIT, or QNFIT in the
SIMFIT package (http://www.simfit.man.ac.uk).
Steady-State Kinetics. The activity during the steady state
was measured spectrophotometrically in 0.1 M sodium phosphate
buffer, pH 7.5, at substrate concentrations ranging from 20 to
250 μM at 22, 30, 38, and 46 °C. A decrease in activity at high
temperatures is expected to be linked to kinetics only, and not to
stability issues, as the t1/2 at 45 °C is >500 min. The substrate was
dissolved in acetonitrile and added to the buffer at a final con-
centration of 1% (v/v) acetonitrile. Enzyme was diluted in 0.1 M
sodium phosphate, pH 7.5, and was added to the reaction mixture
at a final concentration of 400 nM. The rate of uncatalyzed
epoxide hydrolysis was below the detection limit during the assays.
Kinetic parameters kcat and KM were extracted after fitting the
Michaelis-Menten equation by nonlinear regression (MMFIT)
to the experimental data. For the parameters kcat, K0.5, and nH
A kinetic mechanism explaining the formation of both the
(
1S,2R), 3, and (1R,2S), 4, diols produced in StEH1-catalyzed
transformation of 2 is shown in Scheme 1. The model includes
different interconverting forms of the substrate-free enzyme as
well as the Michaelis complexes. The distinct alkyl enzyme
intermediates, resulting from attack at either C-1 or C-2, are
subsequently hydrolyzed into the different diol products.
The enzyme displays a hysteretic behavior with both enantio-
mers of 2-MeSO, as seen in the pre-steady-state measurements,
which indicates the existence of different enzyme forms (12). The
conformational changes coupled to the hysteretic behavior are
supported by theoretical studies on the murine EH; MD simula-
tions of a 4 ns time span following formation of the Michaelis
complex show anticorrelated motion of the lid and core domains
across the active site during catalysis (41).
Regio- and enantioselectivity are closely linked components of
epoxide hydrolase catalysis, both influencing the stereochemical
purity of the product. Substrate selectivity in enzyme catalysis, be
it structural or enantio- or regioselectivity, is determined by
interplaying factors intrinsic to the reacting system. The enan-
tioselectivity is also affected by external conditions such as
temperature, pH, pressure, and composition of the reaction
medium (42, 43). In this work, we have studied the regioselectivity
of StEH1 in greater detail by examining how regioselectivity is
affected by changes in temperature and pH. Kinetic studies in the
steady-state and the pre-steady-state phases, in combination with
(
the Hill coefficient) a rational function with a floating exponent
was fitted to the initial rate data determined at 22 and 30 °C for 2
using INRATE.
The derived steady-state rate law for formation of the sum of
diol products, following the mechanism in Scheme 1, is described
by a second-order expression in the form of eq 1 (44).
2
i½Sꢁ þ j½Sꢁ
v
0
¼
ð1Þ
2
k þ l½Sꢁ þ m½Sꢁ
An evaluation of the individual parameters of eq 1 is presented in
the Supporting Information.
Steady-State Parameters for Formation of Individual
Diols. The steady-state kinetics of StEH1-catalyzed hydrolysis of
2 was followed at 22 and 38 °C, pH of 7.5, in the presence of
135-760 μM epoxide. The concentration for the sum of diols
formed was first determined spectrophotometrically, in triplicate.
Reactions were terminated with the addition of methanol to a

Article
Biochemistry, Vol. 49, No. 10, 2010 2299
Table 1: Steady-State and Pre-Steady-State Parameters for StEH1-Catalyzed Hydrolysis of 2 at pH 7.5
MM equation Hill equation
(mM)
pre steady state
-1
-1
-1
-1
a
-1
cat/K0.5 (s mM
-1
b
H
-1
T (°C)
k
cat (s
)
K
M
k
cat/K
M
(s mM
)
k
cat (s
)
K
0.5 (mM)
k
)
n
k
5
þ k-5 (s
)
22
30
38
46
6.9 ( 3
1.6 ( 0.8
0.49 ( 0.1
0.61 ( 0.2
1.2 ( 0.6
4.3 ( 3
1.4 ( 0.2
3.0 ( 0.6
0.17 ( 0.03
0.22 ( 0.08
8.2 ( 2
14 ( 6
1.7 ( 0.2
1.2 ( 0.2
(1)
6.7 ( 0.8
12 ( 2
51 ( 3
66 ( 5
4.7 ( 0.7
4.8 ( 1
10 ( 5
9.7 ( 2
7.9 ( 3
8.3 ( 6
(1)
a
b
0 M
The substrate concentration resulting in v = Vmax/2. K0.5 = K if n = 1. Hill coefficient.
final concentration of 50%, whereafter solvent was evaporated.
After resolvation of the residue in a 80/20 hexane/2-propanol
mixture, the ratio of each diol was obtained by chiral HPLC, in
duplicate measurements. The total concentration of diols to-
gether with the molar ratios allowed for calculations of the
absolute concentrations of the individual diols at each time point.
Steady-state kinetic parameters were determined by fitting the
Michaelis-Menten equation to the data.
Pre-Steady-State Kinetics. Data were collected with an
Applied Photophysics SX.20MV sequential stopped-flow spec-
trophotometer at different temperatures (22, 30, 38, or 46 °C) in a
0.1 M sodium phosphate solution at pH 7.5. The concentration of
2
was varied between 85 and 750 μM, with an StEH1 concentra-
tion of 10 μM. The substrate was dissolved in acetonitrile before
being added to the reaction mixture, resulting in a final acetoni-
trile concentration of 1% (v/v). The catalyzed reaction was
followed as a decay in the intrinsic tryptophan fluorescence,
excitation λ 290 nm, collecting the emitted light after passage
through a 320 nm cutoff filter. Pre-steady-state kinetic para-
meters were determined from multiple-turnover experiments
under pseudo-first-order conditions, with a minimum of two
repetitions of eight fluorescence traces being collected, all of
which were averaged for the different substrate concentrations.
The apparent rates, kobs, were determined by fitting a single
exponential function with a floating end point, f = A exp(-kobst)
þ C, where f is the averaged fluorescent trace, A the amplitude,
t the time, and C the floating end point. Values of the rate sum
F
concentrations of epoxide 2 in StEH1-catalyzed hydrolysis at
22 °C, pH 7.5. The Michaelis-Menten equation (dashed line) and
a Hill equation (solid line), with a Hill coefficient of 1.7, were fitted to
IGURE 2: Initial steady-state velocities as a function of varying
the data. Error bars represent standard deviations, n g 3.
Difference in Enthalpic and Entropic Contributions to
the Activation Energy. The difference in activation enthalpy
and entropy of the different transition states of the two pathways
leading to the formation of the two product enantiomers was
calculated from
q
q
q
ΔΔG ¼ ΔG
3
-ΔG
4
¼ -RT ln E
ð2Þ
(
k þ k ) (Scheme 1) were obtained after fitting experimental
5
-5
values to eq SI-1 (see Supporting Information) (12, 45, 46).
Regioselectivity. The distribution of diol products was
analyzed by chiral HPLC. Hydrolysis of 2 was conducted at
different temperatures (14, 22, 30, 38, or 46 °C) in 50 mM sodium
phosphate buffer adjusted to pH values ranging from pH 6.0 to
pH 9.0. The reaction mixtures contained a final concentration of
‡
where ΔΔG is the free energy of activation, R the universal gas
constant, T the absolute temperature, and
q
q
q
ΔΔG ¼ ΔΔH -TΔΔS
Combining (2) and (3) allows for extraction of the equation
ð3Þ
1
% (v/v) acetonitrile in a total volume of 1 mL. In the enzyme-
3
4
q
q
lnððkcat=K
M
Þ =ðkcat=K
M
Þ Þ ¼ -ðΔ3 -4ΔH Þ=ðRTÞ þ Δ3 -4ΔS =R
ð4Þ
catalyzed reactions a concentration of 0.1 μM StEH1 was used,
with a concentration of 2 of 0.185 mM. Reactions were termi-
nated by adding methanol to a final concentration of 50% (v/v).
In all enzymatic reactions, at all temperatures and pH values, the
products from the nonenzymatic reactions constituted less than
where Δ3-4ΔH is defined as the difference in activation enthalpy
and Δ3-4ΔS the difference in activation entropy for the product
enantiomers.
1% of the total product yield. The nonenzymatic reactions were
conducted with 14.5 mM 2 in a 50 mM sodium phosphate buffer
and 3% (v/v) acetonitrile. The reactions were terminated at a time
period suitable for determination of the quantitative relationship
of both diols (24-48 h). Following hydrolysis, water was
evaporated from the reaction mixture. The residue was dissolved
in 200 μL of hexane/2-propanol (85/15). The diols, dissolved in
organic solvent, were separated by chiral HPLC. The enzymatic
reactions were performed in duplicate while the nonenzymatic
reactions were performed once. The HPLC analyses were re-
peated twice for all reactions.
RESULTS AND DISCUSSION
Hysteretic Cooperativity. Steady-state kinetics of the cata-
lyzed hydrolysis of 2 (Figure 1) was analyzed at temperatures
ranging from 22 to 46 °C, at pH 7.5. The reaction product is the
sum of diols formed following attack at either of the epoxide
carbons and, thus, does not resolve the individual rates of
formation of the different enantiomers. At 22 and 30 °C the
substrate dependence of the initial velocities showed poor fits to
the Michaelis-Menten model, instead displaying a cooperative

2
300 Biochemistry, Vol. 49, No. 10, 2010
Lindberg et al.
F
IGURE 3: Ratio of the phenylpropyl-1,2-diols 3 and 4 as a function of temperature and pH, expressed in (A) [3]/[4] after the noncatalyzed
3 4
M M
reactions and (B) the ratio of the specificity constants, (kcat/K ) /(kcat/K ) , of StEH1-catalyzed hydrolysis of 2.
behavior (Figure 2, Table 1). As a result, the Michaelis constants
could not be determined accurately and were replaced by an
arbitrary constant, K0.5, defined as the substrate concentration at
Vmax/2. At 30 °C, cooperativity was less pronounced. At the
higher temperatures, cooperativity was completely absent, and
the reactions were well modeled by the Michaelis-Menten
constants in Scheme 1, of each parameter in eqs 5 and 6 is
provided in the Supporting Information.
This behavior is in correspondence to our experimental results.
One of the prerequisites for hysteretic cooperativity, as defined by
Neet and Ainslie, is that the slow transition must be of a rate
lower or as low as the maximum velocity of the system (47). As
the Hill coefficient decreases from a maximum value of 1.7 at
22 °C (pH 7.5) to no observable cooperativity at 38 °C, the sum of
equation, with K0.5 ꢂ K . The values of the individual kinetic
M
parameters kcat and K increased in a compensatory manner
0.5
0
with increasing temperature, resulting in essentially unaltered
catalytic efficiencies (Table 1).
rate constants for interconversion between the ES and E S
complexes (k þ k ) seen from the pre-steady-state measure-
5
-5
Cooperativity is linked to changes in enzyme structure and is
common in allosterically regulated enzymes. There has been no
earlier report on observed cooperative behavior among epoxide
hydrolases, however, although authors have reported on devia-
tions from Michaelis-Menten kinetics in steady-state measure-
ments (25). Cooperativity in monomeric enzymes may arise from
the presence of slow reaction steps on the reaction pathway. In
this context “slow” means “on the same time scale as kcat”. On the
basis of results from pre-steady-state kinetic analysis of catalyzed
hydrolysis of the two enantiomers of trans-2-MeSO we pre-
viously proposed that StEH1 undergoes slow conformational
changes induced by the substrate (Scheme 1). The link between
cooperativity in monomeric enzymes and the concept of hystere-
tic enzymes with slow conformational changes has been estab-
lished. The term hysteretic cooperativity has been coined to
distinguish this from cooperativity resulting from changes in the
quaternary structure of multimeric allosteric enzymes. Requiring
the presence of the two states of free enzyme or enzyme-ligand
complexes, as depicted in Scheme 1, cooperativity arises as a
result of a shift of the dominant pathway with increasing
substrate concentrations. The presence of both hysteretic and
cooperative behavior is not necessarily linked and depends on the
actual values of the rate constants (47, 48).
ments increases with increasing temperature (Table 1). We
propose that this gradually lesser contribution of the rates
0
between the Michaelis complexes ES and E S (k þ k ) to kcat
5
-5
with increasing temperatures is the reason why hysteretic co-
operativity is lost with increasing temperatures. At lower tem-
peratures, the value of k þ k is comparable to the value of kcat
;
5
-5
i.e., at this temperature either k or k can potentially constitute
5
-5
a rate-limiting step for one pathway leading to a specific product
diol. As the sum of k þ k increases with temperature, these
5
-5
rates would become less influential on the resulting kcat.
Catalytic Efficiency and Regioselectivity. The initial rates
of formation of the individual product diols from hydrolysis of 2
were determined. Measurements were performed in the presence
or absence of StEH1 (Supporting Information Tables SI-1 and
SI-2) in a pH range of 6.0-9.0, within a temperature interval of
1
4-46 °C.
In the nonenzymatic hydrolysis the ratios of diols were
compared over the pH and temperature interval (Figure 3A). If
assuming an inversion of configuration takes place as a conse-
quence of an S 2-type reaction, the general trend is an increased
N
preference for ring opening at the aliphatic carbon (30, 49). At a
low pH and higher temperature this results in an essentially
racemic mixture of products. Higher pH values and lower
temperatures direct ring opening toward the benzylic carbon;
at pH 9.0 and 14 °C the enantiomeric excess of 3 is 86%,
corresponding to a molar ratio of 13:1. This is in accordance
with results from other groups, at intermediate temperatures (28
and 30 °C), both in a buffered solution and in water (23, 37).
For the enzymatic reactions the dependence on pH and
temperature is considerably different. The ratios of the specificity
From the derived rate law for Scheme 1 (eq 1), it can be
deduced that if the value of k increases to a large value, the
-
5
steady-state rate equation is simplified to become
2
i½Sꢁ
v0
¼
ð5Þ
2
l½Sꢁ þ m½Sꢁ
i.e.
3
4
constants for each diol, (kcat/K ) /(k /K ) , under these con-
i½Sꢁ
M
cat
M
v
0
¼
ð6Þ
ditions are shown in Figure 3B. In general, the overall sensitivity
to changes in pH and temperature is less pronounced as
compared to the nonenzymatic reaction. Neutral pH conditions
and higher temperatures favor ring opening at the benzylic C-1,
whereas at a pH g8.5 C-2 is preferentially attacked.
l½Sꢁ þ m
Dividing numerator and denominator by l results in the
Michaelis-Menten equation with kcat defined by i/l and KM by
m/l. The dependence, in terms of the individual microscopic rate

Article
To further resolve the individual contributions of the pathways
Biochemistry, Vol. 49, No. 10, 2010 2301
cannot be excluded at this point. The absence of an acid titration
can also be an effect of that the acid-dependent reaction step in
leading to each of the diol products, the kinetics of formation of
the individual diol enantiomers were followed for a chosen set of
temperatures at pH 7.5. A comparison of the kcat values for
formation of each individual diol product (Table 2) with those
determined for the combined formation of both diols (Table 1)
reveals that, at this pH, the factors contributing to increased
selectivity for C-1, to produce diol 3, result in a lowered KM value
at the higher assay temperatures. This results in an overall higher
value of kcat/KM for formation of that diol (Table 2).
the pathway leading to diol 3 is not rate limiting for kcat/K in the
m
reaction leading to diol 4.
We propose that in the two pathways leading to the formation
of diols 3 and 4, the substrate is positioned in two different
binding modes. Theoretical calculations support the supposedly
different binding modes on the related human soluble epoxide
hydrolase (HssEH). The largest structural differences found
between the potato and human enzymes are located in the lid
domains. The two active sites differ somewhat in size and shape
of the cavities in the vicinity of the respective Asp nucleophiles,
but the differences are mainly found at the outer margins of these
cavities (50). When Hopmann and Himo positioned the 2-MeSO
substrate in the active site of HssEH, aiming to create a local
energy minimum to facilitate nucleophilic attack at C-2 in the
initial alkyl enzyme forming step, this required a rearrangement
of the substrate pose in the active site cavity as compared to a
reaction involving attack at C-1. Furthermore, a 180° reposition-
ing of the substrate phenyl group to facilitate attack at either
carbon was almost identical in energy as compared to the original
binding model (51). The same possibility to bind in two opposite
angles in the active site cavity was also observed using the same
substrate in theoretical studies with the murine epoxide hydro-
lase (49).
pH Dependence in Regioselectivity. Plots of the extracted
kcat/KM values against pH (Figure 4) show that the enzyme-
facilitated ring opening at C-1 is dependent on titration of both a
base and an acid, with apparent pK values of approximately 6.7
a
for the conjugate acid of the catalytic base, and 8.2, respectively,
at all temperatures (Table 3). The ring opening at C-2, on the
other hand, is solely dependent on the titration of a catalytic base
to reach maximum activity, with an apparent pKa value of
approximately 6.5 of its conjugate acid. The presence of an acid
titrating with a pK >10 would escape detection in this study and
a
Table 2: Steady-State Kinetic Parameters for StEH1-Catalyzed Formation
of 3 and 4 at pH 7.5
-
1
-1
-1
k
cat (s
)
K
M
(mM)
diol 4
k
cat/K
M
(s mM
)
The possibility of perturbed pK values of catalytic acids has
a
T (°C)
diol 3
diol 4
diol 3
diol 3
diol 4
been addressed in theoretical studies on HssEH and StEH1. The
results proposed that the acid titrated in the basic limb of the pH
profile is the imidazolium form of His300 (StEH1 numbering),
which also serves as the general base of the catalytic triad in its
deprotonated form (51, 52). These investigations give at hand
that the dissociation of the tetrahedral intermediate formed
during hydrolysis of the alkyl enzyme is dependent on a concerted
protonation of the leaving group oxyanion by His300 in its
imidazolium state. Furthermore, based on the kinetic behavior of
an active site mutant enzyme and computer simulations, Tho-
a
a
2
2
8
3.2 ( 0.4 >0.7a 1.5 ( 0.2
>0.5
2.1 ( 0.1 0.89 ( 0.04
5.5 ( 1 1.1 ( 0.6
a
3
2.8 ( 0.3 >0.8 0.52 ( 0.1 >0.5
a
No substrate saturation kinetics within the concentration range used.
a M
Table 3: Apparent pK Values of kcat/K for Formation of Diols 3 and 4 in
StEH1-Catalyzed Hydrolysis of 2 at Different Temperatures
3
4
maeus et al. suggest that these perturbations in pK of His300
a
T (°C)
pKa1
pKa2
pK
a
result from electrostatic changes in the active site microenviron-
ment over the catalytic cycle (52). Assuming that epoxide 2 can be
positioned in two different positions within the active site, this
can be expected to influence the electrostatics in a analogous way
as a consequence to differences in interaction patterns within the
active site in the different pathways leading to the two product
14
22
30
38
46
7.0 ( 0.3
6.8 ( 0.04
6.7 ( 0.3
6.4 ( 0.2
6.5 ( 0.3
8.1 ( 0.3
8.2 ( 0.2
8.1 ( 0.4
8.6 ( 0.2
8.2 ( 0.4
6.4 ( 0.2
6.6 ( 0.1
6.5 ( 0.2
6.6 ( 0.2
6.0 ( 0.2
F
correspond to titrations involving two (A) or one (B) ionization event.
M
IGURE 4: Effect of pH on kcat/K for StEH1-catalyzed formation of 3 (A) or 4 (B) from epoxide 2, at different temperatures. Fitted lines

2
302 Biochemistry, Vol. 49, No. 10, 2010
Lindberg et al.
an increase in difference in activation entropy contribution with
increasing pH (data not shown). This results in free energies of
q
-1
activation, ΔΔG , ranging from -0.8 kJ mol at pH 6.0 to -6 kJ
-
1
mol at pH 9.0. Hence, for the noncatalyzed reaction, the
increased ratio of formation of 3 is primarily dependent on
entropic factors favoring formation of the corresponding transi-
tion states.
The large errors associated with the thermodynamic para-
meters at pH <7.0 are caused by a nonlinear behavior in the
Eyring plot (Supporting Information Figure SI-2) of ln ((kcat/
3
4
K ) /(k /K ) ) versus 1/T . Below 30 °C the slope of the line
M
cat
M
-
1
corresponds to an activation enthalpy of -10 ( 3 kJ mol
accompanied by a large negative activation entropy value of
,
-
1
-1
-
25 ( 10 J K mol . Above 30 °C the slope of the line of the
3
least-squares fit is inversed, with the activation enthalpy changing
-
1
sign to reach a value of 13 ( 2 kJ mol while the entropy
-
1
-1
contribution is positive with a value of 50 ( 6 J K mol . The
3
resulting free energies of activation at 30 °C are negative with
-
1
F
and Gibbs free energy in the reaction pathways leading to formation
IGURE 5: Differences in activation enthalpy, activation entropy,
values corresponding to -2.4 and -1.8 kJ mol , respectively. It
has been suggested by Cainelli et al. that nonlinear Eyring plots of
ln E vs 1/T can result from two different substrate-solvent
clusters (54). As a structural shift between clusters takes place, the
plots are inversed. In the present case Tinv takes place at 30 °C.
Since the Eyring plots for the noncatalyzed reactions do not show
this behavior, it may suggest that temperature-dependent con-
formational changes in the enzyme are the underlying cause for
the nonlinearity.
of the different diol enantiomers 3 or 4. Inset: Linear correlation of
the difference in activation enthalpy versus activation entropy, R =
2
0
.95.
enantiomers. These differences in binding modes are supported
by the simulations with the HssEH where the altered positions of
the substrate resulted in a different hydrogen-bonding pattern
between substrate and enzyme (51).
The slight depression in the ratio of kcat/KM values seen at pH
.0, as compared to neighboring pH values (Figure 3B), cannot
presently be simply explained. The matrix of experimental values
of kcat/KM for each product enantiomer (spanning pH 6.0-9.0 in
one dimension and 14-46 °C in the other) has been repeated with
Hysteresis, Cooperativity, and Promiscuous Regioselec-
tivity. The rate-limiting step in StEH1-catalyzed hydrolysis of
epoxides is in almost all examined cases alkyl enzyme hydro-
lysis (3, 12). However, with epoxide 2 as substrate this has not
been firmly established as the slow structural interconversions
7
0
0
3
50 μM 2, yielding the same experimental pattern (data not
shown).
Thermodynamics in Regioselectivity. Differences in acti-
between E and E and/or ES and E S (Scheme 1) are rate-limiting
for alkyl enzyme formation (12). There are still no available
experimentally determined structures with substrates bound in
the active site, and none of the available crystal structures
supports the presence of conformational changes affecting cata-
lysis in epoxide hydrolases. However, Mowbray et al. reported
on an adaptation of two segments at the “outside” of the active
site cavity between different StEH1 proteins in the same unit cell
resulting from binding of different ligands to the active sites (50).
The segments involved in StEH1 overlap with the structural parts
proposed by Shøitt to be in motion during catalysis of 1 in the
murine epoxide hydrolase (41). Based on our presented results
and the work by Shøitt and Mowbray et al., it is tempting to
ponder on a linkage between regioselective promiscuity and slow
conformational changes seen by the hysteretic kinetic behavior.
The presence of conformational changes is supported by the fact
that the differences in activation enthalpies are positive at all pH,
as would be expected if the highest energy barrier would
constitute a conformational step slower than all other steps in
the catalytic cycle, i.e., hysteresis (47). We propose that these
conformational changes are coupled to alternative binding
modes of the epoxide substrate within the active site.
q
q
vation enthalpy, Δ3-4ΔH , and activation entropy, Δ ΔS ,
3-4
3
derived from the ratios of the kinetic constant (k /K ) /(kcat/
K ) , provide information on differences in catalytic steps and/or
cat
M
4
M
binding events. As stated before, StEH1 preferentially promotes
attack at C-1 at pH values below 8.5, with activation entropy
being the main contributor to the difference in activation energy
(
Figure 5). The enthalpy penalty is compensated by a positive
entropy change (inset in Figure 5) which correlates linearly with
2
an R value of 0.95. The temperature dependences of activation
enthalpy and entropy are similar but work in opposite directions.
This causes a shift in preference toward attack at C-2 at pH g8.5,
as the difference in activation enthalpy becomes the main
contributor to the free energy differences. The temperature where
the enzyme shows no selectivity, defined as the “reversal”
temperature T , is obtained by dividing the activation enthalpy,
r
q
q
Δ3-4ΔH , with the activation entropy, Δ ΔS (53). T increases
3-4
r
with increasing pH (Supporting Information Table SI-5) in the
StEH1-catalyzed hydrolysis of 2. There are no simple relation-
ships between low T values (at low pH) and high regioselectivity,
r
confirming that regioselectivity is a complex function influenced
by many factors.
In the noncatalyzed hydrolysis reactions the differences in
activation enthalpy and entropy contributions in the transition
states of the different reaction pathways differ from the enzyme-
catalyzed reactions. A modified Eyring plot of ln([3]/[4]) versus
In our present working model of the kinetic mechanism we
propose that free StEH1 exists in two different conformers in
solution, interconverting with an equilibrium distribution de-
pending on the values of k and k-0. The existence of both
0
conformers will be observed in the pre-steady-state kinetics if the
substrate can bind productively to both conformers and the
subsequent reaction steps proceed through both pathways and
1/T results in a very small favorable negative difference in
0
activation enthalpy over the whole pH range, accompanied by
are being diverged from ES to E S (or the other way around) with

Article
rate(s) (k 5 and/or k ) lower or close to k values. Regioselective
promiscuity is a direct result of a conversion of a substrate into
two product diols, as seen with epoxide 2, but it does not
necessarily need to be linked to hysteresis. However, from the
negative substrate dependence observed in the pre-steady-state
measurements with 2 it can be concluded that, in this case,
hysteresis and regioselective promiscuity appear linked. Further-
Biochemistry, Vol. 49, No. 10, 2010 2303
Verschueren, K. H. G., and Goldman, A. (1992) The alpha/beta
hydrolase fold. Protein Eng. 5, 197–211.
. Elfstr o€ m, L. T., and Widersten, M. (2005) Catalysis of potato epoxide
hydrolase, StEH1. Biochem. J. 390, 633–640.
. Tzeng, H. F., Laughlin, L. T., and Armstrong, R. N. (1998) Semi-
functional site-specific mutants affecting the hydrolytic half-reaction
of microsomal epoxide hydrolase. Biochemistry 37, 2905–2911.
. Rink, R., Kingma, J., Lutje Spelberg, J. H., and Janssen, D. B. (2000)
Tyrosine residues serve as proton donor in the catalytic mechanism of
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5
cat
3
4
5
6
more, if the sum of k and k is lower or equal to kcat, it is likely
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5
0
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that substrates traversing through E S h ES will show hysteretic
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cooperativity in steady-state measurements.
CONCLUSIONS
7. Moussou, P., Archelas, A., Baratti, J., and Furstoss, R. (1998)
Microbiological transformations. Part 39: Determination of the
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hydrolases: a theoretical analysis and a new method for its determina-
tion. Tetrahedron: Asymmetry 9, 1539–1547.
We have shown that in promiscuous regioselectivity the pre-
ference of which carbon to attack can be manipulated more than
5-fold by altering the temperature and pH. A straightforward test
8
. Faber, K., and Kroutil, W. (2002) Stereoselectivity in biocatalytic
enantioconvergent reactions and a computer program for its deter-
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of the temperature dependence on the product outcome, at two
different pH values, provides a simple method to optimize
reaction conditions in order to reach maximal product purity.
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9. Moussou, P., Archelas, A., Furstoss, R., and Baratti, J. C. (2000)
Clues for the existence of two different epoxide hydrolase activities
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and 4 we propose that the causes for a high degree of
420.
regioselectivity are dependent on the positioning of the substrate
within the active site cavity and that isomerization between these
different conformers involves a conformational change in the
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simulation work (13, 40) and the thermodynamic data presented
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of both enantiomers. Also, lower temperatures lead to relative
increases in formation of diol 4 as compared to higher tempera-
tures. During the later years there has been a growing awareness
about the importance of dynamics in enzymatic catalysis, sup-
ported by both experiments and theoretical studies (55-60). The
presence of conformational changes during the formation of the
diols would suggest that dynamic properties of the enzyme can
play an important role in enzyme-catalyzed hydrolysis of certain
epoxides and contribute to the degree of regioselectivity.
1
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In an earlier study on the hydrolysis of epoxide 2 we proposed
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behavior was observed in the pre-steady-state kinetics. With this
work we show that the same model includes the explanation of
the promiscuous regioselectivity and hysteretic cooperativity.
15. Bellucci, G., Chiappe, C., Cordoni, A., and Marioni, F. (1994)
Different enantioselectivity and regioselectivity of the cytosolic and
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1
1
1
6. Hanzlik, R. P., Edelman, M., Michaely, W. J., and Scott, G. (1976)
18
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18
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ACKNOWLEDGMENT
The authors thank Mikael Holm for contributions to the
project.
1
2
2
9. Kroutil, W., Mischitz, M., Plachota, P., and Faber, K. (1996)
Deracemization of (()-cis-2,3-epoxyheptane via enantioconvergent
biocatalytic hydrolysis using Nocardia EH1-epoxide hydrolase. Tet-
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SUPPORTING INFORMATION AVAILABLE
0. Kroutil, W., Mischitz, M., and Faber, K. (1997) Deracemization of
(
The description of the parameters in the derived steady-state
law, the complete equation used for fitting the pre-steady-state
data, the Eyring plots of the difference in activation energy in the
formation of 3 and 4, tables with the kcat/KM or [3]/[4] for
formation of enantiomers, and a table with the resulting thermo-
dynamic data. This material is available free of charge via the
Internet at http://pubs.acs.org.
()-2,3-disubstituted oxiranes via biocatalytic hydrolysis using bac-
terial epoxide hydrolases: kinetics of an enantioconvergent process.
J. Chem. Soc., Perkin Trans. 1, 3629–3636.
1. Laughlin, L. T., Tzeng, H.-F., Lin, S., and Armstrong, R. N. (1998)
Mechanism of microsomal epoxide hydrolase. Semifunctional site-
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37, 2897–2904.
22. Linderman, R. J., Walker, E. A., Haney, C., and Roe., R. M. (1995)
Determination of the regiochemistry of insect epoxide hydrolase
1
8
catalyzed epoxide hydration of juvenile hormone by O-labeling
studies. Tetrahedron 51, 10845–10856.
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