(Received for publication, December 16, 1994)
From the
In order to investigate the involvement of amino acids in the
catalytic mechanism of the soluble epoxide hydrolase, different mutants
of the murine enzyme were produced using the baculovirus expression
system. Our results are consistent with the involvement of Asp-333 and
His-523 in a catalytic mechanism similar to that of other /
hydrolase fold enzymes. Mutation of His-263 to asparagine led to the
loss of approximately half the specific activity compared to wild-type
enzyme. When His-332 was replaced by asparagine, 96.7% of the specific
activity was lost and mutation of the conserved His-523 to glutamine
led to a more dramatic loss of 99.9% of the specific activity. No
activity was detectable after the replacement of Asp-333 by serine.
However, more than 20% of the wild-type activity was retained in an
Asp-333
Asn mutant produced in Spodoptera frugiperda cells. We purified, by affinity chromatography, the wild-type and
the Asp-333
Asn mutant enzymes produced in Trichoplusia ni cells. We labeled these enzymes by incubating them with the
epoxide containing radiolabeled substrate juvenile hormone III (JH
III). The purified Asp-333
Asn mutant bound 6% of the substrate
compared to the wild-type soluble epoxide hydrolase. The mutant also
showed 8% of the specific activity of the wild-type. Preincubation of
the purified Asp-333
Asn mutant at 37 °C (pH 8), however,
led to a complete recovery of activity and to a change of isoelectric
point (pI), both of which are consistent with hydrolysis of Asn-333 to
aspartic acid. This intramolecular hydrolysis of asparagine to aspartic
acid may explain the activity observed in this mutant. Wild-type enzyme
that had been radiolabeled with the substrate was digested with
trypsin. Using reverse phase-high pressure liquid chromatography, we
isolated four radiolabeled peptides of similar polarity. These peptides
were not radiolabeled if the enzyme was preincubated with a selective
competitive inhibitor of soluble epoxide hydrolase 4-fluorochalcone
oxide. This strongly suggested that these peptides contained a
catalytic amino acid. Each peptide was characterized with N-terminal
amino acid sequencing and electrospray mass spectrometry. All four
radiolabeled peptides contained overlapping sequences. The only
aspartic acid present in all four peptides and conserved in all epoxide
hydrolases was Asp-333. These peptides resulted from cleavage at
different trypsin sites and the mass of each was consistent with the
covalent linkage of Asp-333 to the substrate.
Epoxide hydrolases (EC 3.3.2.3) are enzymes that convert
epoxides to diols via addition of water. They have been found in all
mammalian species tested and found in most organs. Based on subcellular
localization and substrate specificity, five distinct enzyme groups
have been defined: 1) microsomal epoxide hydrolase, 2) soluble epoxide
hydrolase (sEH), ()3) cholesterol epoxide hydrolase, 4)
leukotriene A
epoxide hydrolase, and 5) hepoxilin epoxide
hydrolase. In mammals the majority of research has focused on
microsomal epoxide hydrolase and sEH. Induction of hepatic microsomal
epoxide hydrolase in rats and mice by a variety of foreign compounds
suggests an involvement in xenobiotic
metabolism(1, 2, 3, 4, 5, 6, 7) .
The broad spectrum of substrates utilized by sEH also suggests a
detoxifying function(8) . sEH might also participate in the
metabolism of endogenous substrates since sEH hydrolyzes epoxides of
fatty acids more rapidly than many other
substrates(9, 10, 11) .
Microsomal epoxide
hydrolase has been cloned from rat, human, and
rabbit(12, 13, 14) . More recently sEH has
been cloned from rat, mouse, and
human(15, 16, 17) , and also from plants:
potato (Solanum tuberosum) (18) and mouse-eared cress (Arabidobsis thaliana)(19) . Knowledge of these
sequences enabled authors to perform sequence homology
analyses(20, 21, 22) . They noted that
certain regions of the epoxide hydrolases showed significant sequence
similarity to a bacterial haloalkane dehalogenase HLD1(23) .
HLD1 and by inference epoxide hydrolases belong to a group of enzymes
known as the /
hydrolase fold family(24) .
/
hydrolase fold enzymes are hydrolytic enzymes; all have a
catalytic triad consisting of a nucleophile, a histidine, and an acid.
Until recently, the most accepted hypothesis concerning the mechanism
of action of EH was a general base catalysis involving a histidine that
was thought to form a hydroxyl anion by attracting a proton from a
molecule of water. The anion would then attack the oxirane. Recently,
catalytic amino acids of HLD1 have been identified by x-ray
crystallography(25) . Catalysis by HLD1 proceeds by a two-step
mechanism, where the substrate is covalently bound to the enzyme via an
ester bond with Asp-124. The ester bond is then hydrolyzed by a
molecule of water activated by the His-289-Asp-260 pair in the
active site. It is noteworthy that the amino acids which form the
catalytic triad are conserved in all sEH enzymes so far
cloned(22) . Therefore the hypothesis for a similar mechanism
involving an ester intermediate in the mechanism of action of EH seems
more likely than the general base catalysis hypothesis. This new
hypothesis received additional support recently. Low turnover
experiments with H
O provide evidence for this
hypothesis with the microsomal epoxide hydrolase of rat liver (20) . Although trans-stilbene oxide is the most
commonly used substrate with sEH, we recently chose the epoxide
containing substrate juvenile hormone (JH III) in order to test the
hypothesis of a two step mechanism involving an ester intermediate in
the catalysis by sEH(26) . Its characteristics increased our
chance to demonstrate the presence of an ester intermediate during
catalysis. This substrate has a high affinity for sEH(27) ;
moreover, like other trisubstituted epoxides, it is turned over more
slowly than di- and monosubstituted epoxides(27) . After a
brief incubation of radiolabeled JH III with murine sEH, we isolated a
covalently bound substrate enzyme intermediate. Chemical analysis of
this complex revealed that it was an
-hydroxyacyl enzyme (Fig. 1). We also showed that the binding of the substrate to
the enzyme was inhibited by a selective competitive inhibitor of sEH,
4-fluorochalcone oxide.
Figure 1:
Formation of an
-hydroxyacyl intermediate between JH III and
sEH.
The goal of the work described in this paper was to study the mechanism of action of sEH. With knowledge of the amino acids involved in the catalytic mechanism of HLD1, we used site-directed mutagenesis to evaluate the catalytic importance of different amino acids of recombinant murine sEH. Wild-type sEH was radiolabeled with a substrate (JH III) and digested with trypsin, allowing us to isolate radiolabeled peptides from this protein. These peptides are thought to correspond to the hydroxyacyl intermediate. Determination of their partial sequence and molecular mass allowed us to position these peptides within the protein sequence and thus identify a putative catalytic nucleophile in the protein.
Underlined residues have been modified to generate the corresponding mutations and to add or delete restriction sites in order to select for mutants by restriction digest. Restriction site changes were designed to give the same amino acid in the mutant protein as is found in the wild-type protein. All mutants were confirmed by restriction digest and dideoxy sequencing across the mutation site.
Isoelectric focusing (IEF) was performed in an LKB Multiphor electrophoresis apparatus with precast polyacrylamide gels (Ampholine PAGplate, pH 4.0-6.5; Pharmacia Biotech Inc.) using the conditions recommended by the manufacturer. Gels were cooled to 3 °C and focused for 1 h prior to loading. Aliquots of 15 µg of purified protein were loaded in each lane. Focusing was carried out at pH 4.0-6.5 at 2000 V, 25 watts, 25 mA for 2.5 h. Gels were stained with Coomassie Blue, and pH gradients were calibrated using IEF marker samples (Pharmacia). In some cases the gel lanes were sliced into 1-mm sections and incubated overnight in 200 µl of sodium phosphate buffer (100 mM, pH 7.4), and the supernatant assayed for epoxide hydrolase activity.
Radiolabeled peptides were collected and pooled from eight tryptic digests (800 µg total) in order to determine their amino acid sequence and molecular mass. The pooled fractions containing each peptide were concentrated with a Savant Speed-Vac concentrator prior to analysis. Automated Edman degradation with precolumn PTH derivatization was used for peptide sequencing. An Applied Biosystems gas-phase sequenator (Applied Biosystems, Foster City, CA) coupled to an Applied Biosystems model 120A PTH analyzer and M900 data system was employed. Prior to each separate sequence analysis, the chromatographic system was calibrated with PTH-derivative standards.
Figure 2:
Expression of wild-type and mutant sEH in
baculovirus-infected SF21 cells. For SDS-PAGE (A), all lanes
contain 20 µg of cellular protein, and for Western blot (B), all lanes contain 50 ng of protein. sEH is
affinity-purified murine sEH (lane1). Cellular
proteins are from SF21 cells expressing the: His-237 Asn mutant (lane2), His-263
Asn mutant (lane3), His-332
Asn mutant (lane4),
Asp-333
Asn mutant (lane5), Asp-333
Ser mutant (lane6), His-523
Gln mutant (lane7), wild-type (lane8), and
LacZ (lane9).
Figure 3:
Labeling of wild-type and Asp-333
Asn mutant sEHs by the radiolabeled substrate JH III. The proteins (10
µg) were incubated with the radiolabeled JH III. After 3 s of
incubation, the protein was precipitated. The pellets were washed,
dissolved in 5% SDS (w/v), and transferred to counting vials in order
to monitor radioactivity bound to the protein. The same protocol was
followed using BSA as a control. Each value is the mean of two
independent experiments performed in triplicate. Assuming one catalytic
site per monomeric subunit (62,527 Da), this procedure resulted in the
complete labeling of the protein molecules by
substrate.
The Asp-333 Ser
mutant had no activity, whereas the Asp-333
Asn mutant had 8% or
up to 20% wild-type activity when produced in Tn5B1-4 or in SF-21
cells, respectively. A possible explanation for this observation is
that the Asn-333 in the mutant is being intramolecularly hydrolyzed
back to the Asp-333 by activated H
O. To test this latter
hypothesis, we incubated the wild-type and the Asp-333
Asn
mutant proteins at 37 °C at pH 8. After different times of
preincubation, their specific activities were measured. The mutant
recovered activity as a function of preincubation time (Fig. 4);
after 48 h, the recovery of activity was complete compared to the
wild-type. In addition to recovering catalytic activity, the mutant was
able to covalently bind JH III at the same level as the wild-type (not
shown). Furthermore, analytical IEF demonstrated that the pI of the
mutant was altered from a major band at 5.48 and a minor band at 5.44
before preincubation to a major 5.44 band after preincubation, which is
the pI that was determined for the wild-type. IEF gels were also cut
into slices, proteins were eluted by incubation overnight in buffer and
the supernatant assayed for activity. Before preincubation of the
Asp-333
Asn mutant, the major 5.48 band had no catalytic
activity, while the 5.44 band was catalytically active. After
preincubation the 5.44 band had catalytic activity indistinguishable
from the wild-type enzyme.
Figure 4:
Effect of preincubating the wild-type and
the Asp-333 Asn mutant sEHs on the specific activity. Purified
enzymes were preincubated at 37 °C in 100 mM Tris buffer
(pH 8) containing bovine serum albumin (1 mg/ml) and EDTA (3.3
mM). Enzyme concentrations in the preincubation were 16.5 and
38.7 µg/ml for wild-type and mutant, respectively. All values are
the mean of triplicates with bars representing the standard deviation.
Bars do not appear when standard deviations are too
small.
Figure 5:
RP-HPLC elution profiles of peptides
generated by tryptic digest of the murine wild-type sEH. A,
enzyme (100 µg) was incubated with radiolabeled JH III, then
precipitated and digested with trypsin. Fragments were eluted with a
gradient as follows: 100% A from 0 to 2 min, increased to 75% of B over
110 min, an additional increase to 100% B over 3 min, following by 15
min at 100% B (A and B were HO/trifluoroacetic acid
(99.94:0.06, v/v) and H
O/acetonitrile/trifluoroacetic acid
(19.95:80:0.05, v/v/v), respectively). The flow rate was 0.5 ml/min.
The same procedure was used to obtain profile (B), but the
enzyme was preincubated with 4-FCO before addition of the substrate. A, B, C, and D are the peptides
radiolabeled in presence of JH III only (A) or non
radiolabeled in the presence of 4-FCO and JH III (B). Assuming
one catalytic site per monomeric subunit (62,527 Da), 45% of the
original protein was recovered labeled following the above
procedure.
Figure 6: Radiochromatogram of the tryptic digest of the murine wild-type sEH incubated with JH III. Fractions (500 µl) were collected from the RP-HPLC analysis of Fig. 5A. Aliquot (150 µl) of each fraction was transferred to a scintillation vial. All counts were corrected for differential quenching.
Figure 7: Trypsin cleavage sites in the region of Asp-333. A, B, C, and D are the peptides resulting from the tryptic digest at the different sites. The experimentally determined molecular mass of each peptide is indicated and followed in parentheses by the expected molecular mass.
The purpose of this study was to examine the mechanism of sEH
catalysis and, in particular, to identify catalytic amino acids. Based
on sequence homology, epoxide hydrolases have been classified as
members of the /
hydrolase fold
family(20, 21, 22) . A common feature of the
members of this family is the presence of a catalytic triad consisting
of a nucleophile, a histidine and an acid(24) . Recently, x-ray
crystallography revealed that catalysis by HLD1, which belongs to this
family, proceeds by a two-step mechanism. The substrate is covalently
bound to the enzyme via an ester bond with Asp-124, then this ester
bond is hydrolyzed by a molecule of water activated by the pair
His-289-Asp-260(25) . We tested the hypothesis that a
similar mechanism exists for sEH. Using site-directed mutagenesis and
recombinant enzymes, we investigated the possible involvement of
conserved residues in the catalytic mechanism of murine sEH. Of the
nine histidine residues present in the protein, four are unlikely to be
involved in the catalysis since they are either not conserved (position
204 and 419) (22) or are predicted to be in a strong
helix (position 146) or in a strong
strand (position 249). (
)Furthermore, the decrease of activity after mutation of
His-516 of the recombinant rat sEH was not consistent with the
involvement of this amino acid in the catalysis. (
)Therefore, we focused our attention on the histidine
residues in positions 237, 263, 332, and 523. All of these residues are
good candidates for direct involvement in the catalytic event. Indeed,
the fact that they are all next to a glycine may increase their
accessibility. The His-263
Asn mutant retained 52% of wild-type
activity, suggesting that His-263 is not involved in catalysis.
However, this residue is conserved in all EHs (22) , and its
mutation led to a significant loss of specific activity. This suggests
that His-263 may be important for other properties of the protein (i.e. conformation). Since mutation of His-523 led to the most
dramatic reduction in catalytic activity, our results suggest that it
is the general base involved in water activation. We will test the
hypothesis that His-332 is involved in maintaining an active
conformation orienting the active water or in activation of the epoxide
moiety. The loss of activity of the His-332 mutant may be the result of
a conformational change in the vicinity of Asp-333 due to the
replacement of the histidine by an asparagine. Furthermore, this
His-332 is not conserved in all the different EHs(22) . On the
other hand, His-523 is absolutely conserved in all EHs(22) .
This latter residue is, therefore, a more likely candidate for
participation in the activation of H
O in the EH catalytic
mechanism. We are in the process of confirming this hypothesis by
modifying the enzyme with diethylpyrocarbonate, which is selective for
histidine residues.
We recently used JH III, which is a substrate
with a high affinity and a low turnover, to isolate a hydroxyacyl
enzyme intermediate of the murine sEH(26) . Asp-124 is the
residue that binds to the substrate during catalysis by
HLD1(25) . It corresponds to Asp-333 of the murine sEH and is
conserved in all EHs. Two mutants of Asp-333 were produced. Replacement
of this residue by a serine completely abolished activity. This is in
agreement with the involvement of Asp-333 in the catalysis. However,
the Asp-333 Asn mutant had 20% of wild-type activity when
purified from SF21 cells and 8% of wild-type activity when purified
from Tn5B1-4. Interestingly, this latter mutant bound 6% of the
JH III that the wild-type bound. Deamidation of asparagine residues to
aspartic acid residues has been shown in many cases(34) . Such
an event could explain the low but measurable activity of the Asp-333
Asn and the fact that it still binds the substrate. Different
processing of the expressed protein in the two cells lines might
explain the different levels of activity resulting from different rates
of deamidation. Preincubation of the Asp-333
Asn mutant at 37
°C (pH 8) led to a complete recovery of enzyme activity and the
ability to bind covalently to JH III. Furthermore, preincubation
changed the pI of the mutant from 5.48 to 5.44, which is the pI of the
wild-type enzyme. This is consistent with the results expected if an
asparagine is altered to an aspartic acid. Interestingly, the mechanism
of deamidation may occur via a mechanism similar to the one that has
been shown for catalysis by HLD1. Indeed, it is possible to envisage a
mechanism where histidine activates a molecule of water, which will
then attack the amide and hydrolyze it to aspartic acid.
The fact
that we were able to purify the Asp-333 Asn mutant by affinity
chromatography implies that the mutation did not alter the integrity of
the protein. Our affinity chromatography system is based on the
hypothetical binding of the immobilized ligand to a hydrophobic pocket
near the catalytic site but does not involve the catalytic amino acids.
Therefore, it is unlikely that the loss of activity is the result of a
change in conformation. These data also suggest that the Asp-333
Asn mutation blocks catalysis without totally preventing substrate
binding.
We isolated and characterized four peptides from the wild-type enzyme. These four peptides are the result of a tryptic digest at different sites. They overlap and cover the same region of the protein. These peptides contain the residue involved in the ester bond with the substrate since they were each radiolabeled when the enzyme was incubated with substrate. Furthermore, the binding of the substrate and the radiolabeling of the peptides was inhibited in the presence of the specific inhibitor 4-FCO. The presence of Asp-333 in all of them is further indication of its participation in the catalysis. The other aspartic acid residue present in all four peptides (Asp-320) is only conserved in the human sEH(22) .
In
conclusion, our results are consistent with the hypothesis that
catalysis by sEH occurs via a similar mechanism to catalysis by HLD1,
where the substrate and enzyme are covalently bound. Interestingly, it
has been shown recently, that the irreversible inhibition of human
immunodeficiency virus type 2 protease by
1,2-epoxy-3-(p-nitrophenoxy)propane occurs via a covalent
binding between the protein and the inhibitor. This bond involves an
aspartic acid from the protein and the epoxyde moiety of the inhibitor (35) . We also now have strong evidence for the involvement of
the Asp-333-His-523 pair in catalysis by sEH. This pair
corresponds to the pair Asp-124-His-289, which has been shown to
be catalytic in HLD1. Moreover, a similar His-Asp pair of
residues is conserved in all the enzymes belonging to the /
hydrolase fold family(22) . Consequently, if it is possible to
generalize this mechanism to other members of the family, it could
provide a useful tool for radiolabeling enzymes for purification. Our
results might be more important in elucidating the physiological role
of EHs. Despite the fact that these enzymes have been well
characterized(8) , their functions remain unclear. An
understanding of the mechanism of catalysis allows us to design more
effective substrates and/or inhibitors, which will help assess the role
of these enzymes in vivo.