©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reaction Mechanism of

L

-2-Haloacid Dehalogenase of Pseudomonas sp. YL

IDENTIFICATION OF Asp AS THE ACTIVE SITE NUCLEOPHILE BY ^18O INCORPORATION EXPERIMENTS (*)

(Received for publication, March 21, 1995; and in revised form, May 25, 1995 )

Ji-Quan Liu (1) Tatsuo Kurihara (1) Masaru Miyagi (2) Nobuyoshi Esaki (1) Kenji Soda (1)

From the  (1)Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611, Japan and (2)Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Otsu, Shiga 520-21, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 is the active site nucleophile. When the multiple turnover enzyme reaction was carried out in H(2)^18O with L-2-chloropropionate as a substrate, lactate produced was labeled with ^18O. 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(2)^18O, the enzyme was digested with lysyl endopeptidase, and the molecular masses of the peptide fragments formed were measured by an ionspray mass spectrometer. Two ^18O atoms were shown to be incorporated into a hexapeptide, Gly^6-Lys. Tandem mass spectrometric analysis of this peptide revealed that Asp was labeled with two ^18O 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 alpha-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.


INTRODUCTION

L-2-Haloacid dehalogenase (L-DEX) (^1)catalyzes the hydrolytic dehalogenation of L-2-haloacids with inversion of the C(2) 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 alpha-carbon of the substrate.

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 alpha-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 alpha-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.


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 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^14, 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.

We conducted single and multiple turnover enzyme reactions in H(2)^18O 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, ^18O 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 ^18O 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 ^18O. We show in this paper that the reaction proceeds through the Fig. 1A mechanism and that Asp acts as a catalytic nucleophile.


EXPERIMENTAL PROCEDURES

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. H(2)^18O (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-beta-D-galactoside.

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.

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(2)^18O

For a typical single turnover experiment, 200 nmol of the wild-type L-DEX YL in 50 µl of 50 mM Tris-H(2)SO(4) buffer (pH 9.5) was lyophilized. The reaction was initiated by dissolving the dried enzyme in 50 µl of H(2)^18O 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(2)SO(4) (pH 9.0) previously lyophilized were mixed in 50 µl of H(2)^18O and incubated at 30 °C for 24 h. The reaction mixtures were ultrafiltrated, diluted 10-fold with 50% acetonitrile/H(2)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 ^18O-Labeled Wild-type L-DEX YL in HO

Wild-type L-DEX YL was labeled with ^18O under the same conditions as the multiple turnover reaction in H(2)^18O 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(2)SO(4) buffer (pH 9.5), and the recovered enzyme was used as the ^18O-labeled enzyme to catalyze single and multiple turnover reactions in H(2)O according to the same procedures as described above.

Digestion of ^18O-Labeled L-DEX T15 with Lysyl Endopeptidase

10 nmol of lyophilized L-DEX T15, 1 µmol of L-2-chloropropionate, and 2.5 µmol of Tris-H(2)SO(4) (pH 9.0) were mixed in 50 µl of H(2)^18O 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(2)^18O 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.

For detailed analysis of the peptides containing Asp and Asp, the proteolytic digest of the enzyme incubated in H(2)^18O with or without a substrate was applied to a C(18) 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 10^14 molecules/cm^2.

Amino Acid Sequencing

The amino acid sequences of peptides were determined with a fully automated protein sequencer PPSQ-10 (Shimadzu, Kyoto, Japan).


RESULTS

Single and Multiple Turnover Reactions of Wild-type L-DEX YL in H(2)^18O

Under the single turnover conditions, less than 10% D-lactate produced in H(2)^18O contained ^18O (Fig. 2A), whereas under the multiple turnover conditions, more than 95% D-lactate contained ^18O (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.


Figure 2: Ion spray mass spectra of lactate produced with wild-type L-DEX in H(2)^18O. 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 ^18O-Labeled L-DEX YL in HO

After the multiple turnover reaction in H(2)^18O, L-DEX YL was recovered and used as ^18O-labeled L-DEX YL. The enzyme was not inactivated at all by this procedure. Single and multiple turnover reactions were carried out in H(2)O with ^18O-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 ^18O. But under the multiple turnover conditions, ^18O-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.


Figure 3: Ion spray mass spectra of lactate produced with ^18O-labeled L-DEX in normal H(2)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 ^18O 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 H(2)^18O 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 ^18O atoms were incorporated solely into the peptide 6-11, which contains Asp.


Figure 4: Total ion current chromatogram of proteolytic fragments of ^18O-labeled L-DEX. The molecular mass of each peptide is shown in Table 1.





L-DEX T15 was incubated in H(2)^18O 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 ^18O were incorporated into the peptide 6-11 when L-DEX T15 was incubated in H(2)^18O in the presence of L-2-chloropropionate (Fig. 5B). However, ^18O was not incorporated when the enzyme was incubated in the absence of L-2-chloropropionate (Fig. 5A). No ^18O 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).


Figure 5: Ion spray mass spectra of the peptides containing Asp and Asp. PanelsA and B, Gly^6-Lys hexapeptide derived from L-DEX T15 incubated in H(2)^18O with (B) or without (A) substrate. PanelsC and D, Asn-Lys nonapeptide derived from L-DEX T15 incubated in H(2)^18O 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 ^18O in the hexapeptide 6-11. The parent ions of m/z 654.5 and m/z 650.2, corresponding to ^18O-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 ^18O-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 ^18O of solvent water are incorporated into Asp of the enzyme during the dehalogenation reaction.


Figure 6: Tandem MS/MS daughter ion spectra of ^18O-labeled and unlabeled active site peptides (Gly^6-Lys). A, unlabeled peptide (m/z 650.2, the parent ion). B, ^18O-labeled peptide (m/z 654.5, the parent ion).




DISCUSSION

Single and multiple turnover reaction studies conducted in H(2)^18O 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.

Asp 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 ^18O when the reaction is carried out in H(2)^18O. 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 ^18O atoms were incorporated into Asp, both oxygen atoms of the carboxylate group of Asp are equivalent and either can attack the substrate.

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 is completely conserved. This suggests the essential role of Asp and supports our above conclusion.

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 of L-DEX probably acts as a nucleophile to attack the alpha-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 ^18O 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.


Figure 7: Probable reaction mechanism of L-DEX.




FOOTNOTES

*
This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel: 81-774-33-2594; Fax: 81-774-33-1271; soda{at}pclsp2.kuicr.kyoto-u.ac.jp.

^1
The abbreviations used are: L-DEX, L-2-haloacid dehalogenase; L-DEX YL, thermostable L-2-haloacid dehalogenase from Pseudomonas sp. YL; MS, mass spectrometry; LC, liquid chromatography.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.