From the
Haloalkane dehalogenase (DhlA) from Xanthobacter
autotrophicus GJ10 catalyzes the hydrolytic cleavage of
carbon-halogen bonds in a broad range of halogenated aliphatic
compounds. Previous work has shown that Asp
The first step in the degradation of 1,2-dichloroethane by
bacteria is hydrolytic conversion to 2-chloroethanol catalyzed by
haloalkane dehalogenase. The enzyme from Xanthobacter autotrophicus hydrolyzes a broad range of haloalkanes to the corresponding
alcohols, without a requirement for cofactors or oxygen. The sequence
of the gene ( dhlA) that encodes the 310-amino acid enzyme is
known
(1) . Furthermore, the x-ray structure was determined
(2, 3) . The protein is composed of a main domain, which
is formed by an eight-stranded
Two tryptophans that line an internal
cavity of 37 Å
The N-terminal amino acid sequence was
determined by Eurosequence BV, Groningen, using automatic Edman
degradation
(10) with an Applied Biosystems model 477A
sequencer. In the wild-type and mutant dehalogenases produced by
pELA-based expression vectors, the N-terminal sequence of the enzyme is
MVNAIR instead of MINAIR, which reduces the molecular weight of native
DhlA to 34,990.
For determining incorporation of
Whole proteins were
also analyzed by ion spray mass spectrometry
(13) . Proteins
were dissolved in a solution of 1% trifluoroacetic acid in 80% (v/v)
methanol in water. Multiply charged ion mass spectra were recorded in
1-atomic mass unit steps.
Alkylation of Asp
The
possibility that bromide was released stoichiometrically to enzyme by a
large amount of mutant DhlA was checked with 1,2-dibromoethane.
Incubation of 1.2 mM H289Q DhlA with 3 mM
1,2-dibromoethane led to the release of 1.1 ± 0.2 mM
bromide after 30 s and 1.02 ± 0.2 mM bromide after 5
min, as determined by colorimetric analysis. No formation of
2-bromoethanol could be detected by gas chromatography (detection
limit, 0.1 mM), while the concentration of 1,2-dibromoethane
had decreased by 31%. The data indicate that after the first
stoichiometric release of bromide, the enzyme activity was completely
blocked and that the covalent intermediate accumulated.
Analysis of H289Q enzyme, which was not incubated with
substrate and dialyzed against water, showed the presence of multiply
protonated ions with the number of positive charges varying from 18 to
42 (Fig. 2 A). N-terminal sequencing of the enzyme showed
that 80% of the mutant H289Q haloalkane dehalogenase molecules had lost
the N-terminal methionine. The molecular mass was calculated from the
observed m/ z values of multiply charged ions
(13) . The experimentally determined molecular mass was 34,990
(±2), which is identical to the molecular mass predicted from
the amino acid sequence of H289Q DhlA. The mass of H289Q DhlA that had
reacted with 1,2-dibromoethane was determined in the same way
(Fig. 2 B). A value of 35,100 (±2) was found,
which is 110 mass units higher than the mass of the unreacted mutant
protein. This is very close to the calculated mass increment upon
incorporation of the bromoethyl group, which is 107.
Strong quenching of
fluorescence was observed after incubation of H289Q DhlA with low
concentrations of 1,2-dibromoethane and 1-bromopropane, indicating that
Trp
To determine the binding strength of alkylated
enzyme and halide, H289Q enzyme incubated as above with 1-bromopropane
was extensively dialyzed against TEMAG buffer, pH 7.5. Colorimetric
analysis indicated that the dialyzed enzyme contained less than 10% (on
a molar basis) bromide. Fluorescence quenching persisted, however. Both
at pH 6 and pH 8, the addition of bromide to the diluted dialyzed
protein (1 µM) caused an increase in fluorescence with
typical ligand binding profiles and K
No exchange of the oxygens of the nucleophilic
Asp
We have investigated the role of histidine 289 in the
hydrolysis of bromoalkanes by haloalkane dehalogenase. The properties
of the H289Q mutant show that His
His
Although the position of the histidine residue in the
The role of His
Formation and trapping of the covalent
intermediate formed from DhlA and bromoalkanes appeared to cause
quenching of tryptophan fluorescence, which did not disappear when the
enzyme was dialyzed against buffer to remove excess substrate and free
bromide, indicating self-quenching of tryptophan fluorescence of the
alkyl-enzyme. The addition of bromide to dialyzed alkyl-enzyme caused a
slight increase in fluorescence, which can be explained by bromide
binding to the alkylated protein and a less efficient quenching of
tryptophan in the bromide-bound enzyme than in the bromide-free
alkylated H289Q DhlA.
The dissociation constant of bromide with the
alkylated mutant enzyme at pH 8 was determined to be more than
1000-fold lower than the dissociation constant of bromide with the
nonalkylated wild-type enzyme. Furthermore, the nonalkylated H289Q
enzyme had a very low affinity for bromide, even at pH 6. These
observations indicate that protonation of His
, which is
located close to the internal substrate-binding cavity, carries out a
nucleophilic attack on the C-
of the alkylhalide, displacing the
halogen. The resulting alkyl-enzyme intermediate is subsequently
hydrolyzed. In order to study the role of His
in the
hydrolysis of the intermediate, a His
Gln mutant
was constructed by site-directed mutagenesis. The purified mutant
enzyme was not catalytically active with haloalkanes, but a halide
burst stoichiometric to the amount of enzyme was observed with
1,2-dibromoethane. Using ion spray mass spectrometry, accumulation of
the covalent alkyl-enzyme and binding of the alkyl moiety of the
substrate to an Asp
-containing tryptic peptide were
shown. Fluorescence-quenching experiments indicated that halide ions
are strongly bound by the alkyl-enzyme but not by the substrate-free
enzyme. The results show that His
is the base catalyst
for the dealkylation of the covalent intermediate, but that it is not
essential for the initial nucleophilic attack of Asp
on
the C-1 atom of the haloalkane. Furthermore, the halide ion that is
released in the first step probably leaves the active site only after
hydrolysis of the alkyl-enzyme.
-sheet and
-helices, and a cap
domain, which is formed by helices and loops located on top of the main
domain. On the basis of the topology of the main domain, haloalkane
dehalogenase was classified as a member of the
/
hydrolase
fold enzymes
(4) . This group of hydrolytic proteins have a
conserved arrangement of active site residues in the main domain,
forming a catalytic triad.
, which is the substrate binding site,
bind the halogen atom of the substrate and halide ions
(5, 6) . Substrate and halide binding can be followed by
quenching of tryptophan fluorescence. On the basis of x-ray
crystallographic studies
(5, 6) and mass spectrometric
analysis of
O incorporation from
H
O
(7) , Asp
was proposed
to act as a nucleophile, causing displacement of the halide ion and
formation of an alkyl-enzyme intermediate. The aspartate is located on
the nucleophile elbow, a conserved structural element present in the
main domain of
/
hydrolase fold enzymes
(4) . The
alkyl-enzyme ester bond of the covalent intermediate was proposed to be
hydrolyzed by water. A water molecule could be activated by
His
, which is also at a conserved position in the
/
hydrolase fold structure and may act as a proton acceptor
(Fig. 1). The ``charge relay'' residue that activates
His
is Asp
, which is also present at a
conserved position in these enzymes.
Figure 1:
Proposed
catalytic mechanism of haloalkane dehalogenase. A,
nucleophilic attack of Asp leading to formation of the
covalent alkyl-enzyme intermediate. B, His
catalyzed hydrolysis of the intermediate. The oxygen atom of the
water molecule is incorporated in the carboxylate group of
Asp
. Main chain amide protons serve as oxyanion hole. See
Introduction for details.
In this study, we analyze the
properties of a His
Gln mutant and show that the
histidine is essential for hydrolysis of the covalent intermediate but
not for its formation. Mutation of His
and alkylation of
Asp
were also found to influence the affinity of the
enzyme for halide ions and the rate of substrate-independent exchange
of the carboxylate oxygens of Asp
with solvent water.
Materials
All reagents were purchased from Merck
or Sigma. Restriction enzymes and other molecular biology enzymes were
from Boehringer Mannheim. -Chymotrypsin and
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin were obtained from Worthington Biochemical Corp., NJ. All DNA
primers were purchased from Eurosequence BV, Groningen. DNA sequencing
was done with the T7 sequencing kit from Pharmacia Biotech Inc.
H
O (97%) was obtained from Isotec Inc.,
Miamisburg, OH.
Construction of the Mutant H289Q
Mutants were
constructed by two consecutive PCR(
)
reactions.
Wild-type dhlA DNA was amplified with the mutant primer
5`-GCGGACGCTGGCCAGTTCGTACA (Gln codon underlined) and the
antisense primer located downstream of the dhlA gene
5`-ATAGAATTCATGGATCCTCAGTTTTCGTACCGGCACCGG ( BamHI
site underlined). The PCR product was elongated by a subsequent PCR
reaction with primer 5`-AACCCCTCGAGATAGCGGACCC ( XhoI
site underlined) and the above downstream primer. The new PCR product
was used as mutant DNA and exchanged with the corresponding fragment of
a derivative of the haloalkane dehalogenase expression vector pPJ123
(8) , which contained an additional XhoI site in the
dhlA gene,
(
)
and finally recloned in the
much better DhlA expression vector pELA
(8) . Sequences were
confirmed by T7 DNA polymerase dideoxy sequencing
(9) .
Expression and Purification of Haloalkane
Dehalogenase
Wild-type and mutant H289Q haloalkane dehalogenase
were purified from Escherichia coli BL21(DE3) as described by
Schanstra et al.(8) . Cells with pELA constructs
expressing haloalkane dehalogenase were grown at 30 °C in LB medium
containing 50 µg/ml ampicillin to an A of
about 1. Cultivation was continued at 17 °C, and IPTG (0.4
mM) was added after 1 h as an inducer for DhlA expression.
Cells were harvested after 16 h ( A
2), washed
with TEMAG buffer (25 mM Tris
SO
, pH 7.5,
containing 1 mM EDTA, 1 mM
-mercaptoethanol, 1
mM sodium azide, and 10% (v/v) glycerol), resuspended in TEMAG
buffer, and sonicated for 10 s/ml cell suspension. Cell debris and
other nonsoluble material were removed by centrifugation at 120,000
g for 60 min. This was followed by DEAE-cellulose
chromatography and hydroxylapatite chromatography to yield pure enzyme
(8) . All enzyme samples used were more than 99% pure as
determined by SDS-polyacrylamide gel electrophoresis. Stocks of the
enzyme were stored in TEMAG buffer. For incubations with
H
O, the enzyme was concentrated by
ultrafiltration over an Amicon PM10 filter to 10-20 mg/ml.
Stoichiometric bromide release from 1,2-dibromoethane was tested with
enzyme concentrated to 42 mg/ml.
Assays
Measurements of haloalkane dehalogenase
activity with different substrates were performed by following halide
liberation with a colorimetric assay as described previously
(1) . The assay is done in 1 N nitric acid and thus
results in denaturation of the enzyme. Bromide release from
1,2-dibromoethane by a high concentration of DhlA was tested in
incubation mixtures containing 1.2 mM enzyme, 3 mM
1,2-dibromoethane, and 50 mM TrisSO
, pH 8.2,
in a total volume of 500 µl. Halide liberation was measured
colorimetrically after 30 s and 5 min of incubation at 30 °C.
Calculations were corrected for the absorbance caused by enzyme, using
a control incubation containing no substrate. Protein concentrations
were determined with Coomassie Brilliant Blue or by measuring the
absorbance at 280 nm.
Fluorescence Measurements
Fluorescence spectra and
fluorescence quenching by halide or substrate were determined with an
Aminco SPF500-C spectrofluorometer at 25 °C as described previously
(5) . All incubations were done using 1 µM
dehalogenase in TEMAG buffer with an excitation wavelength of 290 nm.
Emission spectra were recorded from 300 to 400 nm.
Incubations with
H
Incubations of dehalogenase
with HO
O were carried out in 100 µl of
incubation mixtures containing 0.1 mM dehalogenase, 50
mM Tris
SO
, pH 8.2, and 48 or 83%
H
O for the wild-type and H289Q DhlA,
respectively. After 2.5 min or 3 h at 30 °C, 14 µl of 2
M ammonium acetate/ammonium carbonate, pH 8.0, and 5 µl of
1 mg/ml trypsin, freshly dissolved in 0.2 M of the same
buffer, were added. Digestion was carried out for 4 h at 37 °C.
Controls were performed similarly but with 1,2-dibromoethane and
1-bromopropane omitted.
HPLC Isolation of the Peptide Fragments
Trypsin
cleavage was carried out with 5-20 mg/ml haloalkane dehalogenase
and 0.05-0.2 mg/ml trypsin in 0.2 M ammonium
acetate/ammonium bicarbonate buffer, pH 8. After 4 h at 37 °C, the
mixture was separated by reversed phase HPLC on a Nucleosil 10C18
column, using a linear gradient of 0-67% acetonitrile in 0.1%
trifluoroacetic acid as the eluant. Eluting peptides were either
subjected directly to mass spectrometric analysis or collected,
lyophilized, and used for further experiments. The alkylated mutant
enzyme was significantly less sensitive to trypsin digestion, and
therefore a 4-fold higher trypsin concentration and an incubation time
of 20 h were used.
O
from [
O]H
O in Asp
,
trypsin cleavage was done as described above, and fragments were
isolated by HPLC using 0-67% acetonitrile in 0.1%
ammoniumacetate, pH 6, as the solvent instead of trifluoroacetic acid.
The 24-amino acid peptide containing the nucleophilic aspartate
(Asn
-Arg
) was subsequently digested
by chymotrypsin, and a pentapeptide
(Val
-Trp
) containing Asp
was isolated by HPLC as described before
(7) . Control
experiments and previous work
(7) showed that no significant
exchange of carboxylate oxygens with solvent water oxygen occurred
after trypsin digestion of the dehalogenase. The C-terminal carboxylate
of the 24-mer is cleaved off during chemotrypsin digestion.
Ion Spray Mass Spectrometry
For mass spectrometry,
lyophilized peptides were dissolved at approximately 100 nmol/ml in a
solution of 0.02% trifluoroacetic acid in 80% (v/v) methanol in water
and analyzed by pneumatically assisted electrospray ionization on a
Nermag R 3010 quadrupole instrument as described previously
(11, 12) . Data acquisition and data reduction took
place by means of the standard Nermag SIDAR software. Each peptide
solution was examined by full scan mass spectra recorded in 1-atomic
mass unit steps in order to confirm the identity of the peptide and to
determine the incorporation of O.
-containing
peptides was determined by direct introduction of the HPLC column
eluate into the mass spectrometer using a Nucleosil 10C18 reversed
phase column for separation and a 0-67% gradient of acetonitrile
in 0.1% trifluoroacetic acid for elution.
Gas Chromatography Analysis
The presence of
2-bromoethanol and 1-bromopropane in dialyzed enzyme solutions was
measured by gas chromatography. The samples were extracted with
diethylether containing 1-bromohexane as the internal standard.
Analysis and fitting of the data were performed as described by
Schanstra et al.(8) , with the modification that for
1-bromopropane the temperature program of the GC started with 3-min
isothermal operation at 30 instead of 45 °C.
Activity of the H289Q Haloalkane
Dehalogenase
Mutant H289Q haloalkane dehalogenase was
constructed by PCR mutagenesis, expressed in E. coli BL21(DE3)
and purified. Using incubations containing 0.8 mg/ml of enzyme (22
µM) and 5 mM substrate at pH 8.2, no catalytic
activity was found with 1,2-dibromoethane and dibromomethane (detection
limit 0.006 units/mg protein). The wild-type enzyme has an activity
with 1,2-dibromoethane of 4 units/mg of protein under these conditions.
Thus, the activity of the mutant was reduced more than 660-fold.
Identification of the Covalent Alkyl-Enzyme
To
determine whether a covalent alkyl-enzyme did indeed accumulate during
incubation of H289Q DhlA with bromoalkane, whole enzyme and a 24-mer
peptide containing Asp were analyzed by ion spray mass
spectrometry.
Figure 2:
Partial ion spray mass spectra of the
H289Q haloalkane dehalogenase incubated without ( A) and with
( B) 1,2-dibromoethane as substrate. Both spectra were obtained
with an injection of 20 µl of a 10 nmol/ml protein solution.
Multiply protonated protein ion series in A are observed from
m/z 834 (42 charges) through m/z 1944 (18 charges)
and in B from m/z 837 (42 charges) through 1951 (18
charges).
The
nucleophilic aspartate (Asp) of DhlA is present in a
24-amino acid tryptic peptide that can be isolated by reversed phase
HPLC
(7) . To show that this peptide is alkylated during
reaction of H289Q DhlA with 1,2-dibromoethane, the enzyme was incubated
with substrate, digested with trypsin and analyzed by
HPLC/mass-spectrometry (Fig. 3). The peak corresponding to the
24-amino acid peptide (mass, 2601 Da) was detected in trypsin-cleaved
H289Q enzyme that was not exposed to a bromoalkane but was no longer
present in enzyme reacted with 1,2-dibromoethane or 1-bromopropane (
and Fig. 3). Instead, peaks with longer retention
times were detected after trypsin cleavage of enzyme reacted with the
alkylbromides. Mass spectrometry showed the presence of fragments of
2643, 3140, and 4210 Da (accuracy ± 1 Da) in enzyme reacted with
1-bromopropane. This corresponds to the 24-mer
Asn
-Arg
, the 28-mer
Leu
-Arg
, and the 37-mer
Asn
-Arg
, each with a propyl group
attached. Analysis of fragments obtained by trypsin cleavage of H289Q
enzyme incubated with 1,2-dibromoethane revealed the presence of the
same peptides as obtained from the 1-bromopropane-incubated protein but
with a molecular mass that was 65 Da higher, which corresponds to the
difference between the propyl and bromoethyl groups.
Figure 3:
Reversed-phase HPLC elution profiles of
proteolytic fragments. A, trypsin digest of haloalkane
dehalogenase prepared from the unreacted mutant protein. Peak1, 24-mer Asn-Arg
, as
determined by N-terminal amino acid sequencing and mass spectrometry.
B, trypsin digest of H289Q DhlA after reaction with
1-bromopropane. The mass of peaks2-4 was
determined and corresponded to propyl derivatives (see Table I).
C, trypsin digest of H289Q DhlA after reaction with
1,2-dibromoethane. The alkylated peaks are indicated, and their masses
and identities are given in Table I.
These results
confirm that incubation of H289Q DhlA with substrate leads to
accumulation of an alkyl-enzyme with the alkyl group covalently
attached to Asp.
Fluorescence Quenching Experiments
Fluorescence
measurements were used to investigate halide and substrate binding to
H289Q dehalogenase. The binding of halides to wild-type haloalkane
dehalogenase was previously determined by quenching of fluorescence of
the two tryptophan residues that form the halide binding site
(5) . At pH 8, wild-type enzyme bound chloride
(5) and
bromide ions () with Kvalues
of 57 and 5.2 mM, respectively. Dissociation constants for
chloride could not be determined with mutant H289Q DhlA at pH 8 or pH 6
since quenching was very low at concentrations up to 800 mM.
At pH 8, significant quenching of fluorescence of H289Q DhlA was also
not detected with 200 mM bromide, a concentration above which
it became an aspecific collisional quencher. Some quenching was
observed with bromide at pH 6. The degree of quenching was only 15%, as
compared with 30% with the wild-type, and the K
was 4.2 ± 0.7 mM (). This indicates
that binding of bromide and chloride to the H289Q mutant was very poor
as compared with the wild-type enzyme.
and Trp
could still bind substrate
and/or bromide after alkylation (Fig. 4). Quenching with
1,2-dibromoethane was somewhat stronger than with 1-bromopropane.
Dialysis of enzyme incubated with 1,2-dibromoethane or 1-bromopropane
against TEMAG buffer or water did not abolish quenching of
fluorescence, indicating that the quenching is caused either by
covalent modification or by very tight binding.
Figure 4:
Fluorescence spectra of mutant H289Q
haloalkane dehalogenase and quenching by 1,2-dibromoethane or
1-bromopropane. In all measurements, 1 µM mutant enzyme
was used in TEMAG buffer. a, fluorescence spectrum of free
H289Q DhlA; b, fluorescence in the presence of 0.5 mM
1-bromopropane; c, as b, after 48 h of dialysis
against TEMAG buffer; d, as c, after addition of 40
mM KBr; e, fluorescence spectrum of H289Q DhlA in the
presence of 0.5 mM 1,2-dibromoethane; f, as
d, after dialysis.
Theoretically, this
quenching of fluorescence of bromoalkane-exposed and dialyzed H289Q
enzyme could be caused by a bromide ion that did not leave the active
site cavity after cleavage of the carbon-bromine bond by self quenching
of the two tryptophans caused by the covalently attached alkyl group or
by a second substrate molecule that becomes bound to the free
tryptophans of the alkyl-enzyme intermediate after release of the
halide. To determine whether a bromopropane molecule was present in the
active site, 0.93 mM H289Q DhlA was incubated with 5
mM 1-bromopropane for 10 min at 30 °C and pH 8.2 and then
dialyzed for 48 h against TEMAG. The mixture was extracted with
diethylether. No bromopropane could be detected by GC analysis
(detection limit, 0.01 mM). Furthermore, titration of H289Q
DhlA with 1-bromopropane indicated that the quenching of fluorescence
was maximal after addition of an equimolar amount of 1-bromopropane
(Fig. 5). These data suggest that the persistence of quenching
upon dialysis is caused by a tightly bound bromide ion or by the
covalently attached alkyl group, and not by excess substrate bound to
the enzyme.
Figure 5:
Complete quenching of fluorescence of
H289Q mutant haloalkane dehalogenase by a stoichiometric amount of
1-bromopropane. The enzyme concentration used in each measurement was 1
µM, and the fluorescence was measured after addition of
varying concentrations of 1-bromopropane. Fluorescence was measured at
= 346 nm and is given relative to the
unquenched fluorescence of the H289Q mutant
enzyme.
To determine whether halide ions remained bound to the
enzyme after hydrolysis of the C-Br bond during formation of the
covalent adduct, 0.4 mM enzyme was incubated with 0.4
mM 1-bromopropane for 10 min at 30 °C. Subsequently,
enzyme and buffer were separated by centrifugation on a Centricon-10
microconcentrator membrane (cutoff, 10,000). Halide assays revealed the
presence of 0.15 mM bromide in the dialysate. Exact
determination of the amount of bromide bound to enzyme was not possible
due to the high background caused by the protein, but it was estimated
that 0.8 ± 0.2 mM bromide was present in the enzyme
fraction that contained 1.6 mM dehalogenase. Thus, halide ions
were not completely released from the alkylated enzyme into the
surrounding medium.
values that were much lower than those of wild-type or free H289Q
mutant DhlA (). These observations are in agreement with
quenching of fluorescence of alkylated enzyme being caused by the alkyl
group of the bound substrate and/or self quenching of the two
tryptophan residues. No excimer fluorescence emission peak at higher
wavelength (350-450 nm) was detected with the bromide-free
dialyzed alkylated enzyme. Apparently, the alkylated enzyme binds
bromide ions much more tightly than the substrate-free mutant or
wild-type enzymes (), and the degree of quenching is lower
with a bromide ion present in the active site than in the bromide-free
alkylated H289Q dehalogenase (Fig. 4).
Exchange of Carboxylate Oxygens of Asp
Based on sequence homology,
eukaryotic epoxide hydrolase was proposed to have an overall structure
and reaction mechanism similar to haloalkane dehalogenase. Lacourciere
and Armstrong
(14) have incubated epoxide hydrolase under
single turnover conditions with substrate and
Hwith Solvent Water
O, dialyzed the enzyme, and reincubated it
with substrate in the absence of H
O. This led
to incorporation of
O in the second product. Similar
experiments performed by us with haloalkane dehalogenase failed since
there was too much substrate-independent exchange of the Asp
carboxylate oxygens of the intact wild-type enzyme with solvent
water during dialysis (data not shown). The role of His
in this exchange reaction was investigated by comparing the rates
of
O incorporation in the absence of substrate between
wild-type DhlA and H289Q DhlA. The exchange was stopped by trypsin
digestion and only occurred in intact enzyme. Pentapeptides
(Val
-Trp
) containing Asp
were subsequently isolated by HPLC and analyzed by ion spray mass
spectroscopy to identify exchange of the carboxylate oxygens that had
occurred in the presence of H
O
(I).
of mutant H289Q haloalkane dehalogenase was detected
after 3 h of incubation in the presence of H
O,
as the mass of the pentapeptide was predominantly 646. In wild-type
enzyme, however, incorporation of approximately 30%
O
occurred after 3 h of incubation, indicating a rate of exchange
exceeding 0.3 h
.
O incorporation was
hardly observed when wild-type enzyme was incubated for only 2.5 min
(I). Apparently, His
is involved in the
substrate-independent exchange of the carbonyl oxygens of Asp
in the wild-type enzyme. The exchange in the wild-type
dehalogenase in the absence of substrate occurs at a low rate compared
with the reaction rate and rate of incorporation of
O from
solvent of wild-type haloalkane dehalogenase in the presence of
substrate. The exchange can be caused by a His
-bound
water molecule that is observed in the x-ray structure and thus appears
to be highly nucleophilic.
is essential for the
hydrolysis of the covalent alkyl-enzyme intermediate, which is formed
by the nucleophilic attack by Asp
and not for its
formation. Using mass spectrometry, we showed that the intermediate
accumulated when the mutant was incubated with 1-bromopropane or
1,2-dibromoethane. We could not detect any catalytic activity of the
mutant, which indicates that the first order rate constant for
hydrolysis of the covalently alkylated enzyme must be below 3.5
10
s
. For the wild-type enzyme,
this rate constant must be higher than the k
,
which is 2.3 s
.
of haloalkane
dehalogenase is part of a catalytic triad, which is also present in
other
/
hydrolase fold enzymes. The catalytic amino acids are
present along the primary sequence in the order of nucleophile,
aspartate (or glutamate), histidine. Histidine is also completely
conserved in the catalytic triad of serine proteases of the
chymotrypsin class and the subtilisin class, where the three residues
are located along the sequence in the order of histidine, aspartate,
nucleophile and aspartate, histidine, nucleophile, respectively. In the
serine proteases, the histidine is needed both for formation and
hydrolysis of the acyl-enzyme, since it activates the nucleophile by
base catalysis and is also able to donate a proton to the serine
leaving group when the ester is hydrolyzed. The role of His
in DhlA is probably to increase the nucleophilicity of a water
molecule that is close to the carbonyl carbon of Asp
in
the covalent intermediate in the x-ray structure
(3) . The
proton abstracted by the imidazole N-
may be transferred to the
alcohol that is released during hydrolysis of the intermediate.
-sheet
thus is conserved in other
/
hydrolase fold enzymes
(4) , its role in catalysis is very different between members of
this group. In dienelactone hydrolase from Pseudomonas B13,
the equivalent His
is only involved in maintaining the
nucleophilicity of Cys
and not hydrolysis of the
covalently bound intermediate
(15) . In lipase from
Geotrichum candidum and acetylcholinesterase from Torpedo
californica, the histidine is suggested to act as it does in
serine proteases, i.e. it is involved both in formation and in
hydrolysis of the covalent acyl-enzyme intermediate
(16, 17, 18) . For wheat carboxypeptidase A, the
catalytic histidine was also suggested to act as an acceptor of a
proton from the nucleophilic serine
(19) . Thus, the role of the
general base histidine varies in the different
/
hydrolase
fold enzymes. It is noteworthy that hydrolysis of the ester
intermediate by DhlA occurs by nucleophilic attack of this water
molecule on the carbonyl carbon of Asp
(7) and
not on the carbon from which the first leaving group is displaced, as
in the
/
hydrolase fold enzymes that have a serine as the
nucleophile. In the latter enzymes, the carbonyl function essential for
hydrolysis of the substrate and the ester intermediate is provided by
the substrate rather than by the enzyme.
in hydrolysis of the alkyl-enzyme intermediate is based on the
enhancement of the nucleophilicity of a water molecule. In the x-ray
structure, the presence of a water nearby the carboxylate carbon of
Asp
was indeed observed
(5) . His
not only facilitates the cleavage of the alkyl-enzyme but also
causes an unusually rapid exchange of the carboxylate oxygens of
Asp
with oxygen from water. This exchange explains why we
never observed transfer of
O via enzyme to the second
product when DhlA was incubated with substrate in
H
O, dialyzed, and subsequently incubated in
unlabeled water with a second substrate. Such a transfer was recently
observed with microsomal epoxide hydrolase
(14) . This enzyme
has sequence similarity to DhlA, suggesting that the enzymes are
mechanistically similar
(7, 14, 20) . The
nucleophilicity of the water molecule toward Asp in epoxide hydrolase
thus could be lower than in haloalkane dehalogenase. Epoxide hydrolases
also have a conserved histidine residue that aligns with His
of haloalkane dehalogenase
(14) and site-directed
mutagenesis of the corresponding His
of rat microsomal
epoxide hydrolase has indeed shown that this residue is essential for
activity
(21) .
is required
for proper halide binding to the free wild-type enzyme in order to
compensate for the repulsive negative charge of Asp
if
the carboxylate of this residue is not esterified. The strong binding
of bromide to the alkylated enzyme also indicates that the halide ion,
which is the first reaction product, will not leave the active site
until the alkyl-enzyme is hydrolyzed. Probably, halide release occurs
after the alcohol has left the active site cavity since the alcohol was
never observed in x-ray crystallography experiments of enzyme soaked in
substrate, whereas halide was always present
(6) .
Table:
Mass
of peptide fragments of H289Q DhlA
Table:
Fluorescence quenching of dehalogenases by
bromide
Table: 0p4in
Relative abundancies of m/z 646, 650, and 652 expected from 48 and 30%
O
incorporation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.