(Received for publication, April 10, 1995; and in revised form, July 11, 1995)
From the
O-Labeled epoxides of trans-1,3-diphenylpropene oxide (tDPPO) and cis-9,10-epoxystearic acid were synthesized and used to
determine the regioselectivity of sEH. The nucleophilic nature of sEH
catalysis was demonstrated by comparing the enzymatic and nonenzymatic
hydrolysis products of tDPPO. The results from single turnover
experiments with greater or equal molar equivalents of sEH:substrate
were consistent with the existence of a stable intermediate formed by a
nucleophilic amino acid attacking the epoxide group. Tryptic digestion
of sEH previously subjected to multiple turnovers with tDPPO in
H
O resulted in the isolation and purification
of a tryptic fragment containing Asp-333. Electrospray mass
spectrometry of this fragment conclusively illustrated the
incorporation of
O. After complete digestion of the latter
peptide it was shown that Asp-333 of sEH exhibited an increased mass.
The attack by Asp-333 initiates enzymatic activity, leading to the
formation of an
-hydroxyacyl-enzyme intermediate. Hydrolysis of
the acyl-enzyme occurs by the addition of an activated water to the
carbonyl carbon of the ester bond, after which the resultant
tetrahedral intermediate collapses, yielding the active enzyme and the
diol product.
Mammalian epoxide hydrolases (E.C. 3.3.2.3) have been implicated
in the metabolism of epoxide containing xenobiotics, many of which are
believed to be mutagenic and/or
carcinogenic(1, 2, 3) . During the past 20
years the mammalian microsomal epoxide hydrolase (mEH) ()has
received a great deal of attention partly due to its higher selectivity
for cyclic and arene oxides(4, 5, 6) . A
great deal of work has provided a clear picture of the regio-, stereo-,
and enantiospecificity of
mEH(7, 8, 9, 10) . Most researchers
agree that mEH hydrolyzes the epoxide via an anti (commonly
referred to as trans) opening of the oxirane, where attack of
the nucleophile usually takes place at the least sterically hindered
carbon. These results are substantiated by substituent effects on the
rate of hydrolysis as investigated by Dansette et al.(11) and kinetic solvent isotope studies as reported by
Armstrong et al.(12) . By the use of single turnover
experiments in H
O (13) , Lacourciere
and Armstrong have also postulated that the hydrolysis of epoxides by
mEH proceeds via the intermediary of a nucleophilic amino acid,
yielding an acyl-enzyme intermediate, which is hydrolyzed further to
release the diol product and the active enzyme. This is in contrast
with the previously more accepted hypothesis in which an activated
water molecule was thought to be responsible for direct attack on the
epoxide.
Much less work has been completed on sEH, but the
preliminary data closely parallel those for mEH. Because of the
critical role of sEHs in the metabolism of xenobiotics and their
possible involvement in the biosynthesis of
metabolites(14, 15, 16, 18) , ()a detailed understanding of the catalytic mechanism of sEH
is imperative. The anti opening of epoxides has been
demonstrated on a select few substrates along with
H
O studies, which have shown that nucleophilic
attack occurs on the less hindered carbon (7, 19, 20) . Recently, Hammock et al.(21) were able to trap a postulated acyl-enzyme
intermediate, suggesting the possible involvement of a nucleophilic
amino acid in the hydration of epoxides by sEH. Pinot et al.(22) were able to isolate a tryptic fragment of sEH
containing the postulated acylated amino acid. The latter discovery is
very significant in light of studies that have compared the sequence
homology of several EHs. These studies have shown significant sequence
similarity among EHs and a bacterial haloalkane dehalogenase (DhlA,
from the bacterium Xanthobacter autotrophicus), which, among
other regions of homology, possesses a conserved histidine and aspartic
acid tandem(23, 24, 25) . DhlA belongs to a
group of enzymes known as the
/
hydrolase fold family, all of
which contain a catalytic triad consisting of a nucleophile, a
histidine, and an acid(26) . If EHs, and particularly sEH in
our case, belong to the
/
hydrolase fold family of enzymes
then it can be inferred that the conserved aspartic acid is responsible
for the nucleophilic attack on the epoxide, resulting in the
acyl-enzyme intermediate observed by Hammock et
al.(21) .
Herein we detail experiments associated with the mechanism of sEH. Results of regioselectivity of hydration, direction of oxirane opening, mechanism of hydration, and identification of the putative catalytic amino acid are reported. Also, possible mechanisms of inhibitory action by chalcone oxides and glycidols are discussed.
Acidic hydrolyses of the
same epoxides were performed by introducing 2 µl of the substrates
(stock) into 5% perchloric acid solution (100 µl). After 30 min of
incubation at 37 °C in a water bath, saturated NaCl (100 µl)
and enough 10% KOH was added to neutralize the solution. Basic
hydrolysis of O/
O-labeled tDPPO was effected
by the addition of 2 µl of each epoxide to a 10% KOH solution (100
µl) followed by incubation for 20 h at room temperature, at which
time saturated NaCl (100 µL) was added and the reaction mixture was
neutralized with 12 M HCl.
The enzymatic and chemical
hydrolysis products were extracted with similar efficiency by the
addition of 450 µl of ethyl acetate. Each test tube was vortexed
vigorously and centrifuged in order to clearly separate the organic and
aqueous phases. The top layer (ethyl acetate, 400 µl) was withdrawn
and placed in a conical reaction vessel, isooctane (5 µl) was added
as a trap solvent, and the solvent was evaporated under nitrogen flow.
Each sample was derivatized 30 min prior to GC/MS analysis with BSTFA
(50 µl). The latter experiments were performed in quadruplicate.
The enzymatic hydrolyses were performed with three separate enzyme
preparations. It should be noted that [H]tDPPO
was synthesized for substrate activity studies (
)and was
utilized here to measure the efficiency of ethyl acetate extractions of
the epoxide and its corresponding diol. It was found that the ethyl
acetate extracts contained >99.9% of the
[
H]diol and 100% of the
[
H]epoxide that were originally incubated in the
assay buffer.
Figure 1: Reverse phase HPLC chromatograms of tDPPO and its corresponding diols. a, the threo- and erythro-diols of tDPPO generated by acidic hydrolysis of tDPPO. b, threo-diol of tDPPO (threo-1,3-diphenyl-1,2-propanediol) generated by cis-hydroxylation of trans-1,3-diphenylpropene. c, 100 mM tDPPO incubated at 37 °C with 3.9 µg/ml sEH in sodium phosphate buffer (100 mM, pH 7.4) containing 100 µg/ml BSA after 5 min. d, same as panel c after 40 min.
Figure 2: sEH exhibits a high regioselectivity of epoxide opening for tDPPO with over 97% of the attack occurring at the benzylic position. The regioselectivity of sEH was less discriminating in the hydrolysis of cis-9,10-epoxystearic acid and methyl cis 9,10-epoxystearate.
[O]Epoxystearic acid also shows some
regioselectivity of epoxide opening with sEH. Acidic hydrolysis of
[
O]cis-9,10-epoxystearic acid results
in a 1:1 distribution of carbon 9- and carbon 10-labeled diol, a
testament to the chemical equivalence of the two epoxide carbons.
However, as depicted in Fig. 2, 68.5% ± 1.3% of the
enzymatic attack occurs at carbon 10. The
O label
distribution following enzymatic hydrolysis of
[
O] methyl cis-9,10-epoxystearate is
identical with the distribution following enzymatic opening of its
corresponding free acid, suggesting that the anchoring of the
carboxylic acid or ester on the enzyme is not the driving force for the
observed regioselectivity.
Figure 3:
Two proposed schemes for the hydrolysis of
epoxides by sEH. Mechanism 1 presupposes the activation of
water by a histidine residue, which leads to the direct opening of the
epoxide. If performed in HO, the resulting
diol would incorporate
O oxygen after one
turnover. Mechanism 2 presupposes that a nucleophilic amino
acid such as a carboxylate anion opens the epoxide, which would lead to
the formation of an
-hydroxyester-enzyme intermediate.
Subsequently, an activated water hydrolyzes the ester, which releases
the diol product, and the enzyme is regenerated. If performed in
H
O, the resulting diol would not incorporate
O oxygen after one turnover. Acidic
activation of the epoxide oxygen, which could enhance catalytic
activity, is consistent with both
mechanisms.
The latter
result was substantiated further by single turnover hydrolysis and
analysis of cis-9,10-epoxystearic acid in
HO. The K
and V
for epoxy stearic acid and tDPPO were found to
be 11.0 µM and 3460 nmol/min/mg protein, and 2.80
µM and 26200 nmol/min/mg protein, respectively.
Since the oxidized fatty acid turned over more slowly than tDPPO,
a smaller fraction of the enzyme population experienced multiple
turnover. This hypothesis is supported by the better fit of the cis-9,10-epoxystearic acid data to what is expected
theoretically as compared with the data obtained for tDPPO (Fig. 4).
Figure 4:
The graph illustrates single
turnover experiments performed with various ratios of sEH:substrate.
The theoretical line (-
) represents the
theoretical data based on mechanism 2. The data representing
single turnover experiments performed with tDPPO
(
) and cis-9,10-epoxystearic acid
(
) clearly follows the pattern dictated
by mechanism 2. The theoretical line (
-
) represents the theoretical data obtained
based on mechanism 1.
From our previous observations the expected site of O insertion within tDPPO is at the benzylic carbon. The
amount of
O label incorporated in the product was
monitored by GC/MS for each experiment. As can be seen in Fig. 4, equal or greater molar equivalence of sEH (i.e.
1 theoretical turnover) led to minimal
O
incorporation from the H
O solvent. The same
pattern was observed with cis-9,10-epoxystearic acid (Fig. 4). These data suggest
O incorporation from
enzyme onto the epoxide leading to the formation of an acyl-enzyme
intermediate.
Figure 5:
Reverse phase HPLC spectra for the tryptic
digestion of sEH treated with excess tDPPO in
HO. Peaks I-IX were isolated
and concentrated by microbore HPLC (>100 fold) as described under
``Materials and Methods.''
In this study we set out to examine the mechanism of epoxide
hydration by sEH. Previous publications from this and other
laboratories have reported some stereo- and regiochemical controls
exerted by sEH. It is generally accepted that sEH opens epoxides by
nucleophilic attack (as compared with a general acid catalysis) often
at the least hindered carbon in an S2 fashion. This is
normally accomplished by the back side attack of the epoxide carbon (anti opening)(20) . Therefore, the initial
stereochemistry of the epoxide dictates the stereochemical outcome of
the diol product. trans-1,3-Diphenylpropene oxide was
synthesized as a general substrate for sEH. The asymmetry about the
epoxide allows regiochemical studies of nucleophilic attack. Also, the
nature of the epoxide carbons vary greatly, one being benzylic, which
greatly favors nucleophilic attack, and the other being homobenzylic,
which is slightly less hindered. As can be seen from Fig. 1, sEH
opens tDPPO via a back side displacement of the epoxide oxygen. This is
evident from the exclusive production of erythro-diol from the trans-epoxide substrate. A completely acid-catalyzed mechanism
would probably result in a mixture of threo- and erythro-diols since the carbocation involved in the mechanism
would be greatly stabilized by the phenyl group and would allow water
to attack the carbocation from either face due to its trigonal planar
geometry. However, the latter data do not preclude the possibility of
an acid assisted catalysis occurring in concert with a base catalyzed
attack of the epoxide. Transient carbocations can retain their
tetrahedral geometry, and therefore, block one face of nucleophilic
water attack, which would result in the formation of either erythro- or threo-diol (depending on the
stereochemistry of the parent epoxide) and not both.
Next, the
regioselectivity of sEH with tDPPO, cis-9,10-epoxystearic acid
and methyl cis-9,10-epoxystearate was investigated. The
[O]epoxides of the latter substrates were
synthesized as described previously. The chemically (base- and
acid-catalyzed) and enzymatically generated diols were analyzed by
GC/MS to determine label incorporation into each position. In the case
of sEH-hydrolyzed tDPPO, 97.1% of the attack occurred at the benzylic
position (Fig. 2). The same pattern was observed for the
base-hydrolyzed [
O]tDPPO (97.0%). Even though
the homobenzylic carbon is slightly less sterically hindered, the
nucleophilic attack occurs at the benzylic carbon, which can better
stabilize the transition state. As expected, the acid-catalyzed
hydrolysis of [
O]tDPPO led to products that
exclusively exhibited attack of the benzylic position due to the
stability of the benzylic carbocation.
We were also able to show
that sEH is able to discriminate two chemically similar epoxide
carbons. The enzymatic hydrolysis of
[O]cis-9,10-epoxystearic acid led to a
68.5% attack of C-10 in favor of C-9 (Fig. 2). The methyl ester
of [
O]cis-9,10-epoxystearic acid was
also subjected to sEH hydrolysis with identical results, discounting
the importance of the carboxylate anion of this fatty acid being
anchored by the enzyme. The observed selectivity of sEH between the two
nearly identical epoxide carbons can be due to several reasons. The
accessibility of the nucleophilic amino acid to C-9 versus C-10 can account for the observed data. Another possibility is the
different spatial orientations by which the fatty acid can be absorbed
onto the catalytic site. This could lead to a different population of
C-9 versus C-10 positioned closer to the catalytic amino acid
for nucleophilic attack (if one spatial orientation is favored over
others). Of course, the true explanation might be a combination of the
latter two hypotheses. The regioselectivity data were instrumental in
the calculations of single turnover data.
After firmly establishing
the regioselectivity and mode of attack with both tDPPO and cis-9,10-epoxystearic acid, the mechanism of action could be
probed. Two scenarios could be envisioned. The first hypothesis, which
has been the generally accepted theory, alleges an activated water
(possibly by a histidine) delivery onto the epoxide carbon and
subsequent protonation of the generated alkoxide to yield the product (Fig. 3, mechanism 1). The second theory supposes that
the side chain of a nucleophilic amino acid such as a carboxylate anion
within sEH attacks and opens the epoxide. The resultant acyl-enzyme
intermediate would then be hydrolyzed by an activated water (again
postulated via a histidine) that would generate the native enzyme and
release the diol product (Fig. 3, mechanism 2). Even
though there has not been direct evidence for the second postulated
mechanism, recent discoveries from the mechanism of DhlA and single
turnover experiments performed with mEH by Lacourciere and Armstrong (13) raised the possibility that sEH could also follow the same
mechanistic path(25, 35) . X-ray crystallographic
study of DhlA with bound substrate has clearly shown the involvement of
an aspartic acid as the nucleophile, which in the first step of
enzymatic catalysis attacks the substrate leading to the formation of
an acyl-enzyme intermediate (-hydroxyester-enzyme) (36) .
Also, single turnover experiments with mEH incubated in
H
O have shown that the oxygen introduced
within the epoxide is not
O-labeled during the first
turnover. The latter data suggest that mEH also hydrolyzes epoxides via
an acyl-enzyme intermediate(13) .
Fig. 3illustrates
the difference in products which would be obtained from a single
turnover experiment performed in HO based on
the two postulated mechanisms. Since the first mechanism presupposes a
direct attack of an activated water onto the epoxide, it follows that
under any conditions of hydrolysis the
O would be
incorporated within the product. Therefore, after a single turnover of
sEH with tDPPO the diol product should be labeled with
O.
However, if the hydrolysis follows the second mechanism suggested, then
the oxygen incorporated during the first run of the enzyme is supplied
by the enzyme. Since the amino acids of sEH contain only
O
oxygens it is clear that the diol product obtained from the first
turnover would contain only
O oxygen. The labeled oxygen
from water would hydrolyze the acyl-enzyme intermediate and, therefore,
incorporate itself within the enzyme. The enzyme would therefore
contain a 1:1
O:
O labeled amino acid after
one turnover. As multiple turnovers occur, the catalytic amino acid
would be completely labeled with
O and would yield diol
products identical to the first suggested mechanism, hence the
importance of performing single turnover experiments.
As indicated
from the data in Fig. 4, the lack of O within the
diol product for equimolar or greater sEH:substrate refutes the direct
incorporation of
O from H
O into
the epoxide. Care must be taken in such an experiment to insure each
enzyme does not turn over more than one substrate by adding diluted
substrate and mixing enzyme and substrate at cold temperatures. This is
particularly important for substrates turned over with a high k
such as tDPPO. Initially our results were
inconclusive since we added concentrated substrate to the enzyme buffer
solution. Based on our initial results it is safe to assume most of the
substrate was consumed as the mixing occurred within pockets of high
substrate concentration. This would lead to a population of enzyme
experiencing multiple turnovers. Proper mixing resulted in the data in Fig. 4, which are consistent with expected results of the second
mechanism involving the intermediary of an acyl-enzyme.
Results of
single turnover experiments conclusively showed the involvement of a
nucleophilic amino acid within sEH responsible for the
di-O oxygen observed in the product that had been obtained
by hydrolysis of substrate in H
O. With the
recent investigation into the homology of epoxide hydrolase genetic
sequences (23, 24) and their probable evolutionary
connection with the
/
hydrolase fold family of
enzymes(26) , a conserved region containing an aspartic acid at
position 333 of sEH has been identified. Consequently, the probable
catalytic Asp-333 could be labeled with
O with excess
substrate hydrolyzed by sEH in H
O (via
multiple enzyme turnovers that would incorporate
O within
the catalytic amino acid). Tryptic digestion of sEH labeled with
H
O and H
O yielded
identical HPLC elution profiles, and each peak could be separated for
further analysis. Table 1lists the molecular weights obtained
for reverse phase HPLC-purified, microbore HPLC-concentrated tryptic
fragments I-IX by electrospray mass spectrometry for sEH incubated with
excess tDPPO in H
O and
H
O buffer. Peaks I and III did not produce
results that were interpretable in light of the protein sequence. Peaks
II, VI, VII, and VIII in both the H
O- and
H
O-treated groups exhibit molecular weights
identical to those predicted theoretically. These observations indicate
the absence of any labeled oxygen within those fragments. The
difference between fragments VI and VIII is 2 Da, which could be
attributed to disulfide bridge formation within T16. These data suggest
that in the absence of specific catalysis there is no rapid exchange of
water into carboxylic acid functionalities under the conditions used
here.
As can be seen from Table 1, there is an average
increase of 3.6 Da per fragment for peaks IV, V,
V
, IX
, and IX
for the
H
O-treated sEH as compared with the
H
O-treated sEH, which corresponds to almost
two isotopic oxygens substituted per fragment. The molecular mass of
the fragment isolated as peak IV from the
H
O-treated group was 3,218.3 Da, which matches
the expected molecular mass for the tryptic fragment T26 (3218.8 Da).
The T26 fragment isolated from the H
O-treated
sEH increased 3.5 Da, therefore implicating T26 as the fragment
containing the catalytic amino acid. Peaks IV, V
,
V
, IX
, and IX
, which also contain
the labeled oxygen are the result of incomplete tryptic digestion
upstream and downstream from T26. The identity of T26 was further
confirmed by amino acid sequencing of its N terminus (not shown), which
clearly supported our assignment of the fragment. The reason an
increase of 3.6 Da is observed as opposed to 4.0 Da (from the
incorporation of two
O atoms) might be attributed to
experimental error in molecular mass determination. However, close
examination of the MaxEnt-transformed spectra reveals that the peak
widths are broader for the
O-labeled peptides than for the
unlabeled peptides. This suggests the presence of a small amount of
peptide that contains only a single
O, and this would
account for the molecular weights being slightly less than expected for
incorporation of two
O units. It should be noted that the
isotopic purity of the [
O]water used for
experiments was 95% at best.
The presence of two O
units within T26 clearly indicates the involvement of an amino acid in
the delivery and opening of tDPPO. The failure to see any increase in
mass in peaks II, VI, VII, and VIII and the increase in 4 Da rather
that 8 Da or more in peaks IV, V
, V
,
IX
, and IX
support the hypothesis that there is
minimal exchange of H
O with the carboxylic
acids of aspartic and glutamic acids unless there is a catalytic
involvement. Single turnover experiments with sEH preincubated at 37
°C in H
O for 1 h yielded the exact same
results as the normal single turnover experiment, discounting any
catalytic exchange of
O into the catalytic amino acid
without the presence of substrate. The cDNAs and predicted peptide
sequence of T26 contains Asp-333 as the only aspartic acid within the
fragment. As discussed previously, Asp-333 has been implicated as the
catalytic residue by sequence homology to other EHs, and the
/
fold hydrolase family of enzymes. Based on the latter data,
it seems very plausible that Asp-333 is the catalytic amino acid
responsible for the initial step of hydrolysis. However, T26 contains a
glutamic acid (Glu-348), which is not conserved but could conceivably
be responsible for catalytic activity.
The complete digestion of T26
(from both HO and H
O
treated sEH) was achieved with a nonspecific protease (immobilized
protease Sg). After derivatization of the hydrolyzed amino acid
residues with MTBSTFA the samples were analyzed by GC/MS. Since there
is only one aspartic acid in T26, and since the aspartic acid
derivative obtained from the hydrolysis of the
[H
O]T26 exhibited an increased mass (Table 2), it seems unequivocal that the aspartic acid in T26 is
responsible for the nucleophilic attack onto tDPPO. Furthermore, it was
shown that Glu-348 (the only other carboxylate containing amino acid in
T26) did not incorporate any
O oxygen. The attack of
Asp-333 initiates enzymatic activity, leading to the formation of an
-hydroxyester-enzyme intermediate. Hydrolysis of this acyl-enzyme
is accomplished by the addition of an activated water to the carbonyl
carbon of the ester bond, after which the resultant tetrahedral
intermediate collapses, yielding the active enzyme and the diol
product. The second step of the latter mechanism resembles the
mechanism of most serine esterases and proteases.
A push-pull
mechanism, where the epoxide oxygen is activated by protonation or
hydrogen bonding that would weaken the C-O epoxide bond seems
likely since the carboxylate anion (Asp-333) is the attacking
nucleophile. The latter hypothesis fits well with our knowledge of
carboxylate chemistry. Chemically, carboxylates are not considered
strong nucleophiles that could easily open epoxide rings. However, the
epoxide carbon can be chemically activated toward nucleophilic attack
by weak nucleophiles such as carboxylate anions through the
coordination of the epoxide oxygen with a lewis acid(37) .
Within the catalytic cavity of sEH, the epoxide oxygen can be activated
by various amino acids through either proton donation or hydrogen
bonding. The x-ray crystallographic solution of DhlA co-crystallized
with substrate clearly demonstrated that two tryptophan residues are
responsible for polarization of the halide-leaving group(36) .
Genetic sequence homology between DhlA and sEH investigated by Arand et al.(23) has shown that one of the two tryptophans
in DhlA is conserved in sEH. This latter postulated tryptophan
(Trp-334) could activate the epoxide by hydrogen bonding with the
oxirane oxygen, but it is not necessarily the only mechanism for
epoxide activation by sEH. A push-pull mechanism has also been
suggested previously based on the general structure of sEH
inhibitors(34, 38, 39) . The general
structure of sEH inhibitors contains a carbonyl or hydroxy to the
epoxide. It has been suggested that the presence of the
-hydroxy/carbonyl functionality interferes with the acidic
activation of the epoxide by hydrogen bonding with the proton
donor(17) . However, in light of a two-step mechanism of action
a detailed study is necessary to elucidate the inhibitory mechanism.
In conclusion, we report that we have been able to conclusively show that sEH effects hydrolysis of epoxides via an A2 type, back side nucleophilic attack of Asp-333. An isolatable acyl-enzyme intermediate is formed, which is hydrolyzed by an activated water, resulting in the regeneration of the active enzyme and the release of the diol product.