(Received for publication, March 21, 1995; and in revised form, May 25, 1995 )
From the
L-2-Haloacid dehalogenase (EC 3.8.1.2) catalyzes the
hydrolytic dehalogenation of L-2-haloacids to produce the
corresponding D-2-hydroxy acids. We have analyzed the reaction
mechanism of the enzyme from Pseudomonas sp. YL and found that
Asp
L-2-Haloacid dehalogenase (L-DEX) ( We have isolated and purified thermostable L-2-haloacid dehalogenase (L-DEX YL) from a
2-chloroacrylate-utilizable bacterium, Pseudomonas sp.
YL(6, 7) , cloned its gene(8) , and
constructed the overexpression system(9) . The enzyme is
composed of 232 amino acid residues(8) , and its amino acid
sequence is highly similar to those of L-DEXs from other
bacterial strains and haloacetate dehalogenase H-2 from Moraxella sp. strain B; 36-70% of residues are
identical(5, 10, 11, 12, 13, 14) .
Accordingly, these dehalogenase reactions probably proceed through the
same mechanism. Two different mechanisms have been proposed for the
reactions of L-DEXs (Fig. 1)(14) . According to
the mechanism shown in Fig. 1A, a carboxylate group of
Asp or Glu acts as a nucleophile to attack the
Figure 1:
Proposed mechanisms of L-DEX. A, nucleophilic attack by acidic amino acid residue followed
by ester hydrolysis. B, a general base catalytic
mechanism.
Site-directed mutagenesis
experiments have been extensively carried out to elucidate the
catalytic amino acid residues of L-DEX(15, 16, 17, 18) .
His We
conducted single and multiple turnover enzyme reactions in
H
The cells were
collected by centrifugation, suspended in 50 mM potassium
phosphate buffer (pH 7.5), and disrupted by ultrasonic oscillation at 4
°C for 20 min with a Seiko Instruments model 7500 ultrasonic
disintegrator. The cell debris was removed by centrifugation. The
supernatant solution was brought to 40% saturation with ammonium
sulfate, and the precipitate was removed by centrifugation. The
supernatant solution was dialyzed against 2000 volumes of 50 mM potassium phosphate buffer (pH 7.5) for 14 h and subsequently
applied to a DEAE-Toyopearl 650M column. The elution was carried out
with a linear gradient of 50-300 mM potassium phosphate
buffer (pH 7.5). The active fractions were pooled and used as the
purified enzyme.
For detailed analysis of the peptides
containing Asp
Figure 2:
Ion spray mass spectra of lactate produced
with wild-type L-DEX in H
Figure 3:
Ion spray mass spectra of lactate produced
with
Figure 4:
Total ion current chromatogram of
proteolytic fragments of
L-DEX T15 was incubated in
H
Figure 5:
Ion spray mass spectra of the peptides
containing Asp
Figure 6:
Tandem MS/MS daughter ion spectra of
Single and multiple turnover reaction studies conducted in
H Asp Recent computer
analysis revealed that L-DEX belongs to a large superfamily of
hydrolases with diverse specificity (21) . These proteins
include different types of phosphatases and numerous uncharacterized
proteins from eubacteria, eukaryotes, and Archaea. Among all of these
proteins, Asp The same reaction mechanism in which a nucleophilic carboxylate
group takes part has been proposed for three types of hydrolases: rat
liver microsomal epoxide hydrolase(22) , haloalkane
dehalogenase from Xanthobacter autotrophicus GJ10(23) , and (4-chlorobenzoyl)coenzyme A dehalogenase
from Pseudomonas sp. strain CBS3(24) . Epoxide
hydrolase and haloalkane dehalogenase are structurally related to each
other, but (4-chlorobenzoyl)coenzyme A dehalogenase does not share
sequence identity with either of these two enzymes. L-DEX does
not show a significant sequence similarity to any of these three
enzymes. Hence, L-DEX resembles these hydrolases solely by the
presence of an active site nucleophilic carboxylate. In conclusion,
Asp
Figure 7:
Probable reaction mechanism of L-DEX.
is the active site nucleophile. When the multiple
turnover enzyme reaction was carried out in H
O
with L-2-chloropropionate as a substrate, lactate produced was
labeled with
O. However, when the single turnover enzyme
reaction was carried out by use of a large excess of the enzyme, the
product was not labeled. This suggests that an oxygen atom of the
solvent water is first incorporated into the enzyme and then
transferred to the product. After the multiple turnover reaction in
H
O, the enzyme was digested with lysyl
endopeptidase, and the molecular masses of the peptide fragments formed
were measured by an ionspray mass spectrometer. Two
O
atoms were shown to be incorporated into a hexapeptide,
Gly
-Lys
. Tandem mass spectrometric analysis of
this peptide revealed that Asp
was labeled with two
O atoms. Our previous site-directed mutagenesis experiment
showed that the replacement of Asp
led to a significant
loss in the enzyme activity. These results indicate that Asp
acts as a nucleophile on the
-carbon of the substrate
leading to the formation of an ester intermediate, which is hydrolyzed
by nucleophilic attack of a water molecule on the carbonyl carbon atom.
)catalyzes the hydrolytic dehalogenation of L-2-haloacids with inversion of the C
configuration producing the corresponding D-2-hydroxy
acids. The enzymes have been isolated from various bacteria and
characterized(1, 2, 3, 4, 5, 6) .
They have several common properties; their molecular weights are
between 25,000 and 28,000, they show the maximum reactivities in the pH
range of 9-11, they specifically act on the L isomer of a
substrate, their substrates contain a carboxylate group, and the
halogen atom to be released is bound to the
-carbon of the
substrate.
-carbon of L-2-haloacid, leading to the formation of an ester
intermediate. This is hydrolyzed by an attack of the water molecule
activated by a basic amino acid residue of the enzyme. Alternatively,
water is activated by a catalytic base of the enzyme and directly
attacks the
-carbon of L-2-haloacid to displace the
halogen atom (Fig. 1B). In both mechanisms, a
positively charged amino acid residue is suggested to bind the
carboxylate group of the substrate.
of L-DEX from Pseudomonas cepacia MBA4 was proposed to act as a catalytic base as shown in Fig. 1B(15) . However, replacement of
His
of L-DEX YL, corresponding to His
of the P. cepacia enzyme, by a few other residues caused
no inactivation of the enzyme, indicating that this His is not involved
in catalysis(17) . We mutated all the 36 highly conserved
charged and polar amino acid residues of L-DEX YL and found
that the enzyme activity is decreased significantly by replacement of
Asp
, Asp
, Lys
,
Ser
, Arg
, Thr
,
Tyr
, and Asn
(18) . However, we
could not show which mechanism of those shown in Fig. 1is
involved in the reaction and identify the catalytic residue.
O in the present study. The single turnover
reaction was carried out in the solution containing the enzyme in
excess of substrate, whereas the multiple turnover reaction was done by
using an excess amount of substrate. If the reaction proceeds through
the Fig. 1B mechanism,
O is incorporated
into the product both in single and in multiple turnover reactions. In
the case of the reaction through the Fig. 1A mechanism,
the single turnover reaction causes
O incorporation into
the carboxylate group of the enzyme but not into the product. In the
multiple turnover reaction, both the product and the carboxylate group
of the catalytic residue are labeled with
O. We show in
this paper that the reaction proceeds through the Fig. 1A mechanism and that Asp
acts as a catalytic
nucleophile.
Materials
pBA5 encoding L-DEX YL was
constructed as described in the previous paper(8) . The
1.5-kilobase pair PstI-BamHI fragment of pBA5
containing the 3`-noncoding region was removed, and the remaining
3.6-kilobase pair fragment was circularized to form pBA501. Escherichia coli BMH 71-18 mutS, E. coli BW313,
and helper phage M13K07 were purchased from Takara Shuzo (Kyoto,
Japan). DNA modifying enzymes were obtained from Takara Shuzo or Toyobo
(Osaka, Japan). Lysyl endopeptidase of Achromobacter lyticus M497-1 was purchased from Wako Industry Co., Ltd. (Osaka,
Japan). DEAE-Toyopearl 650 M was from Tosoh (Tokyo, Japan). L-2-Chloropropionate was purchased from Sigma.
HO (95-98%) was obtained from Cambridge
Isotope Laboratories (Andover, MA) and Nippon Sanso (Tokyo, Japan). All
other chemicals were of analytical grade.
Introduction of Lysyl Residues into L-DEX YL to
Be Digested by Lysyl Endopeptidase
Lysyl residues were
introduced into the enzyme by site-directed mutagenesis in order to
produce lysyl endopeptidase hydrolytic sites. The template
single-stranded DNA was prepared by infecting recombinant E. coli BW313 carrying plasmid pBA501 with M13K07 phage under the
conditions specified by the manufacturer. Replacement of
Leu, Ser
, and Arg
by Lys was
carried out by the method of Kunkel(19) . The mutant enzymes
and synthetic mutagenic primers are as follows (the underlines indicate
the mutagenized nucleotides): L11K,
5`-ACAGCGTACCGTACTTGTCGAAGGCAATACC-3`; S176K,
5`-GCGTTCTTCGACACGAACAGG-3`; R185K, 5`-GAAGCCGAAGTATTTCGCCCCCG-3`. The
substitutions were confirmed by DNA sequencing with Dye Terminator
sequencing kit and an Applied Biosystem 370A DNA sequencer. The
constructed triple mutant named L-DEX T15 was produced by E. coli BMH 71-18 mutS.
Cultivation of the Cells and Purification of the
Enzyme
Recombinant E. coli cells were cultivated at 37
°C for 14-18 h in Luria-Bertani medium (1% polypeptone, 0.5%
yeast extract, and 1% NaCl, pH 7.0) containing 150 µg/ml of
ampicillin and 0.2 mM
isopropyl-1-thio--D-galactoside.
Enzyme and Protein Assay
L-DEX was
assayed with 25 mML-2-chloropropionate as a
substrate. The chloride ions released were measured
spectrophotometrically according to the method of Iwasaki et
al.(20) . One unit of the enzyme was defined as the amount
of enzyme that catalyzes the dehalogenation of 1 µmol of
substrate/min. Protein assay was done with a Bio-Rad protein assay kit. Single and Multiple Turnover Reactions of Wild-type L-DEX YL in H
For a typical
single turnover experiment, 200 nmol of the wild-type L-DEX YL
in 50 µl of 50 mM Tris-HO
SO
buffer
(pH 9.5) was lyophilized. The reaction was initiated by dissolving the
dried enzyme in 50 µl of H
O containing 20
nmol of L-2-chloropropionate, and the mixture was incubated at
30 °C for 24 h. For a multiple turnover experiment, 10 nmol of L-DEX, 1 µmol of L-2-chloropropionate, and 2.5
µmol of Tris-H
SO
(pH 9.0) previously
lyophilized were mixed in 50 µl of H
O and
incubated at 30 °C for 24 h. The reaction mixtures were
ultrafiltrated, diluted 10-fold with 50% acetonitrile/H
O
(1:1), and then introduced into the mass spectrometer by using a
Harvard Apparatus syringe infusion pump operating at 2 µl/min. The
molecular mass of the produced lactate was measured with a PE-Sciex API III triple quadrupole mass spectrometer equipped with an
ionspray ion source in the negative ion mode (Sciex, Thornhill,
Ontario, Canada).
Single and Multiple Turnover Reactions of
Wild-type L-DEX YL was labeled
with O-Labeled Wild-type L-DEX YL in
H
O
O under the same conditions as the multiple turnover
reaction in H
O by adjusting the molar ratio of
enzyme and substrate to 1:100. The reaction was carried out at 30
°C for 2 h. The reaction mixture was dialyzed against 50
mM Tris-H
SO
buffer (pH 9.5), and the
recovered enzyme was used as the
O-labeled enzyme to
catalyze single and multiple turnover reactions in H
O
according to the same procedures as described above.
Digestion of
10 nmol of lyophilized L-DEX
T15, 1 µmol of L-2-chloropropionate, and 2.5 µmol of
Tris-HO-Labeled L-DEX T15
with Lysyl Endopeptidase
SO
(pH 9.0) were mixed in 50 µl of
H
O and incubated at 30 °C for 24 h. The
protein was denatured with 8 M urea and subsequently digested
at 37 °C for 12 h with 80-100 pmol of lysyl endopeptidase. L-DEX T15 incubated in H
O without
substrate was used as a control.
LC/MS Analysis of the Proteolytic Digest
The
proteolytic digest was loaded onto a packed capillary perfusion column
(Poros II R/H, 320 µm 10 cm LC Packings, San Francisco, CA)
connected to the mass spectrometer and then eluted with a linear
gradient of 0-80% acetonitrile in 0.05% trifluoroacetic acid over
40 min at a flow rate of 10 µl/min. The total ion current
chromatogram was recorded in the single quadrupole mode with a PE-Sciex
API III mass spectrometer equipped with an ionspray ion
source. The quadrupole was scanned from 300 to 2000 atomic mass units
with a step size of 0.25 atomic mass units and a 0.5-ms dwell time per
step. Ion spray voltage was set at 5 kV, and the orifice potential was
80 V. The molecular mass of each peptide was calculated with MacSpec
software supplied by Sciex.
and Asp
, the proteolytic
digest of the enzyme incubated in H
O with or
without a substrate was applied to a C
column (Puresil 5
µ C18 120Å, 4.6
150 mm; Millipore, Tokyo, Japan) and
eluted with 0.05% trifluoroacetic acid for 5 min followed by a linear
gradient of 0-80% acetonitrile in 0.05% trifluoroacetic acid over
60 min at a flow rate of 1.0 ml/min. The elution was monitored at 215
nm with a UV detector, and the fractions were collected and injected
into a PE-Sciex API III mass spectrometer in the single
quadrupole mode under the same conditions as described above.
Identification of the Active Site Peptide by Tandem MS/MS
Spectrometry
The MS/MS daughter ion spectra were obtained in the
triple quadrupole daughter scan mode by selectively introducing the
peptides containing Asp (m/z 654.5 or m/z 650.2) from Q1 into the collision cell (Q2) and
observing the daughter ions in Q3. Q1 was locked on m/z 654.5 or 650.2. Q3 was scanned from 50 to just above the molecular
weight of the peptide. Step size was 0.1, and dwell time was 1 ms/step.
Ion spray voltage was set at 5 kV, and the orifice potential was 100 V.
Collision energy was 30 eV. The resolution of Q1 and Q3 was
approximately 500 and 1500, respectively. The collision gas was argon,
and the gas thickness was 2.9
10
molecules/cm
.
Amino Acid Sequencing
The amino acid sequences of
peptides were determined with a fully automated protein sequencer
PPSQ-10 (Shimadzu, Kyoto, Japan).
Single and Multiple Turnover Reactions of Wild-type L-DEX YL in H
Under the
single turnover conditions, less than 10% D-lactate produced
in HO
O contained
O (Fig. 2A), whereas under the multiple turnover
conditions, more than 95% D-lactate contained
O (Fig. 2B). These suggest that an oxygen atom of water
molecule is first transferred to the enzyme and then to the product.
This supports the mechanism involving an ester intermediate shown in Fig. 1A, but does not the Fig. 1B mechanism, in which an oxygen atom of solvent water is directly
transferred to the product.
O. The
spectra were obtained between 85 and 95 atomic mass units. Step size
was 0.1 atomic mass unit, and dwell time was 10 ms/step. Ion spray
voltage was set at -3.5 kV, and the orifice potential was
-50 V. A, single turnover reaction. B, multiple
turnover reaction.
Single and Multiple Turnover Reactions of
After the multiple turnover reaction in
HO-Labeled L-DEX YL in
H
O
O, L-DEX YL was recovered and used
as
O-labeled L-DEX YL. The enzyme was not
inactivated at all by this procedure. Single and multiple turnover
reactions were carried out in H
O with
O-labeled L-DEX YL. The results are shown in Fig. 3. Under the single turnover conditions (Fig. 3A), more than 90% D-lactate produced
contained
O. But under the multiple turnover conditions,
O-labeled D-lactate was no more than 5% (Fig. 3B). These results also suggest that an oxygen
atom of solvent water is first incorporated into the enzyme, and then
transferred to the product.
O-labeled L-DEX in normal H
O.
The spectra were obtained under the same conditions as described in the Fig. 2legend. A, single turnover reaction. B,
multiple turnover reaction.
Construction, Purification, and Characterization of L-DEX T15
To identify the position of the incorporated O in the enzyme, a mutant enzyme L-DEX T15 was
constructed by introducing three lysyl residues at positions 11, 176,
and 185 of L-DEX YL by site-directed mutagenesis. The
substitutions were confirmed by DNA sequencing. The mutant enzyme was
purified to homogenity by DEAE-Toyopearl column chromatography.
Properties of the mutant L-DEX T15 such as specific activity
toward L-2-chloropropionate and optimum pH were identical to
those of the wild-type enzyme.
LC/MS Analysis of the Peptides Proteolytically Formed
L-DEX T15 was used to carry out a multiple turnover
reaction in HO with L-2-chloropropionate as a substrate. After completion of the
reaction, the enzyme was digested with lysyl endopeptidase, and the
resulting peptide fragments were separated on a capillary column
interfaced with an ionspray mass spectrometer as a detector. When the
spectrometer was in the single quadrupole mode, the total ion current
chromatogram displayed several peaks (Fig. 4). Peaks 1, 2, 3, 4,
5, and 6 were assigned to peptides 109-113, 177-185,
1-5, 6-11, 114-176, and (12-108 +
186-228), respectively (Table 1). Peptides 12-108 and
186-228 are supposed to be bound to each other by a disulfide
bond, though this bond could form artificially because the proteolysis
was carried out under the aerobic condition. The molecular mass of
peptide 6-11 was 654.5 Da, which is approximately 4 Da higher
than the predicted molecular mass (650.75 Da), although the amino acid
sequence of this peptide was Gly-Ile-Ala-Phe-Asp-Lys, which is
identical to that predicted from the nucleotide sequence. Molecular
masses of all other peptides were indistinguishable from the predicted
ones. These results indicate that two
O atoms were
incorporated solely into the peptide 6-11, which contains
Asp
.
O-labeled L-DEX. The
molecular mass of each peptide is shown in Table 1.
O with or without L-2-chloropropionate under the multiple turnover conditions.
The enzyme was digested with lysyl endopeptidase, and the peptides
6-11 and 177-185 containing Asp
and
Asp
, respectively, were isolated with a reverse phase
high performance liquid chromatography column. Two atoms of
O were incorporated into the peptide 6-11 when L-DEX T15 was incubated in H
O in the
presence of L-2-chloropropionate (Fig. 5B).
However,
O was not incorporated when the enzyme was
incubated in the absence of L-2-chloropropionate (Fig. 5A). No
O incorporation was observed
for the peptide 177-185, whether L-DEX T15 was incubated
in the presence or absence of L-2-chloropropionate (Fig. 5, C and D).
and Asp
. PanelsA and B, Gly
-Lys
hexapeptide derived from L-DEX T15 incubated in
H
O with (B) or without (A)
substrate. PanelsC and D,
Asn
-Lys
nonapeptide derived from L-DEX T15 incubated in H
O with (D) or without (C)
substrate.
Analysis of the Active Site Peptide by Tandem MS/MS
Spectrometry
Fragmentations of the peptides were performed using
a mass spectrometer in the daughter ion scan mode in order to determine
the incorporation position of O in the hexapeptide
6-11. The parent ions of m/z 654.5 and m/z 650.2, corresponding to
O-labeled
and unlabeled hexapeptides, respectively, were selected in the first
quadrupole and subjected to collision-induced fragmentation in a
collision cell in the second quadrupole. The daughter ions produced are
shown in Fig. 6. The Y`` series ions at m/z 484.0, 413.1, and 266.0 of
O-labeled peptide
correspond to the fragments of Ala-Phe-Asp-Lys, Phe-Asp-Lys, and
Asp-Lys, respectively. They are about 4 Da higher than those of ions at m/z 480.3, 409.1, and 262.0 of the unlabeled peptide.
However, after the deletion of Asp, molecular masses of the remaining
portions (Lys) of these two peptides were closely similar to each other
(146.8). These results suggest that two atoms of
O of
solvent water are incorporated into Asp
of the enzyme
during the dehalogenation reaction.
O-labeled and unlabeled active site peptides
(Gly
-Lys
). A, unlabeled peptide (m/z 650.2, the parent ion). B,
O-labeled peptide (m/z 654.5, the parent
ion).
O suggested that an oxygen atom from water is
incorporated into the product via incorporation into enzyme. This
observation is not consistent with the general base mechanism shown in Fig. 1B but rather with the Fig. 1A mechanism, where an active site carboxylate functions as a
nucleophile, and an ester intermediate is produced.
and Asp
were shown to be important for catalysis by
our site-directed mutagenesis experiments(18) . Therefore, we
tried to find which residue acts as a nucleophile shown in Fig. 1A. The nucleophilic residue is expected to be
labeled with
O when the reaction is carried out in
H
O. LC/MS and tandem MS/MS analysis showed
that an oxygen atom of solvent water was incorporated into the
carboxylate group of Asp
, but not into Asp
.
Accordingly, the dehalogenation reaction of L-DEX probably
proceeds through the ester intermediate mechanism in which Asp
functions as a nucleophile (Fig. 1A). Since two
O atoms were incorporated into Asp
, both
oxygen atoms of the carboxylate group of Asp
are
equivalent and either can attack the substrate.
is completely conserved. This suggests the
essential role of Asp
and supports our above conclusion.
of L-DEX probably acts as a nucleophile to
attack the
-carbon of the substrate to form an ester intermediate,
which is hydrolyzed by attack of an activated water molecule (Fig. 7). This is the first evidence for the catalytic action of
Asp
in the L-DEX reaction. The combination of
O incorporation experiment and tandem mass spectrometrical
analysis of the labeled enzyme is an effective approach to the
catalytic mechanism of the member of the hydrolase superfamily.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.