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• W2008584836 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 Asp10 is the active site nucleophile. When the multiple turnover enzyme reaction was carried out in H218O 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 H218O, 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, Gly6-Lys11. Tandem mass spectrometric analysis of this peptide revealed that Asp10 was labeled with two 18O atoms. Our previous site-directed mutagenesis experiment showed that the replacement of Asp10 led to a significant loss in the enzyme activity. These results indicate that Asp10 acts as a nucleophile on the α-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. 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 Asp10 is the active site nucleophile. When the multiple turnover enzyme reaction was carried out in H218O 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 H218O, 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, Gly6-Lys11. Tandem mass spectrometric analysis of this peptide revealed that Asp10 was labeled with two 18O atoms. Our previous site-directed mutagenesis experiment showed that the replacement of Asp10 led to a significant loss in the enzyme activity. These results indicate that Asp10 acts as a nucleophile on the α-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. INTRODUCTIONL-2-Haloacid dehalogenase (L-DEX) ( 1The abbreviations used are: L-DEXL-2-haloacid dehalogenaseL-DEX YLthermostable L-2-haloacid dehalogenase from Pseudomonas sp. YLMSmass spectrometryLCliquid chromatography. )catalyzes the hydrolytic dehalogenation of L-2-haloacids with inversion of the C2 configuration producing the corresponding D-2-hydroxy acids. The enzymes have been isolated from various bacteria and characterized(1Goldman P. Milne G.W.A. Keister D.B. J. Biol. Chem. 1968; 243: 428-434Abstract Full Text PDF PubMed Google Scholar, 2Little M. Williams P.A. Eur. J. Biochem. 1971; 21: 99-109Crossref PubMed Scopus (51) Google Scholar, 3Motosugi K. Esaki N. Soda K. Agric. Biol. Chem. 1982; 46: 837-838Google Scholar, 4Tsang J.S.H. Sallis P.J. Bull A.T. Hardman D.J. Arch. Microbiol. 1988; 150: 441-446Crossref Scopus (52) Google Scholar, 5Jones D.H.A. Barth P.T. Byrom D. Thomas C.M. J. Gen. Microbiol. 1992; 138: 675-683Crossref PubMed Scopus (50) Google Scholar, 6Liu J.-Q. Kurihara T. Hasan A.K.M.Q. Nardi-Dei V. Koshikawa H. Esaki N. Soda K. Appl. Environ. Microbiol. 1994; 60: 2389-2393Crossref PubMed Google Scholar). 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 α-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(6Liu J.-Q. Kurihara T. Hasan A.K.M.Q. Nardi-Dei V. Koshikawa H. Esaki N. Soda K. Appl. Environ. Microbiol. 1994; 60: 2389-2393Crossref PubMed Google Scholar, 7Hasan A.K.M.Q. Takada H. Koshikawa H. Liu J.-Q. Kurihara T. Esaki N. Soda K. Biosci. Biotech. Biochem. 1994; 58: 1599-1602Crossref Scopus (28) Google Scholar), cloned its gene(8Nardi-Dei V. Kurihara T. Okamura T. Liu J.-Q. Koshikawa H. Ozaki H. Terashima Y. Esaki N. Soda K. Appl. Environ. Microbiol. 1994; 60: 3375-3380Crossref PubMed Google Scholar), and constructed the overexpression system(9Liu J.-Q. Kurihara T. Nardi-Dei V. Okamura T. Esaki N. Soda K. Biodegradation. 1995; (in press)PubMed Google Scholar). The enzyme is composed of 232 amino acid residues(8Nardi-Dei V. Kurihara T. Okamura T. Liu J.-Q. Koshikawa H. Ozaki H. Terashima Y. Esaki N. Soda K. Appl. Environ. Microbiol. 1994; 60: 3375-3380Crossref PubMed Google Scholar), 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(5Jones D.H.A. Barth P.T. Byrom D. Thomas C.M. J. Gen. Microbiol. 1992; 138: 675-683Crossref PubMed Scopus (50) Google Scholar, 10Kawasaki H. Toyama T. Maeda T. Nishino H. Tonomura K. Biosci. Biotech. Biochem. 1994; 58: 160-163Crossref PubMed Scopus (41) Google Scholar, 11Schneider B. Muller R. Frank R. Lingens F. J. Bacteriol. 1991; 173: 1530-1535Crossref PubMed Google Scholar, 12van der Ploeg J. van Hall G. Janssen D.B. J. Bacteriol. 1991; 173: 7925-7933Crossref PubMed Google Scholar, 13Murdiyatmo U. Asmara W. Tsang J.S.H. Baines A.J. Bull A.T. Hardman D.J. Biochem. J. 1992; 284: 87-93Crossref PubMed Scopus (50) Google Scholar, 14Kawasaki H. Tsuda K. Matsushita I. Tonomura K. J. Gen. Microbiol. 1992; 138: 1317-1323Crossref PubMed Scopus (86) Google Scholar). Accordingly, these dehalogenase reactions probably proceed through the same mechanism.Two different mechanisms have been proposed for the reactions of L-DEXs (Fig. 1)(14Kawasaki H. Tsuda K. Matsushita I. Tonomura K. J. Gen. Microbiol. 1992; 138: 1317-1323Crossref PubMed Scopus (86) Google Scholar). According to the mechanism shown in Fig. 1A, a carboxylate group of Asp or Glu acts as a nucleophile to attack the α-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 α-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.Site-directed mutagenesis experiments have been extensively carried out to elucidate the catalytic amino acid residues of L-DEX(15Asmara W. Murdiyatmo U. Baines A.J. Bull A.T. Hardman D.J. Biochem. J. 1993; 292: 69-74Crossref PubMed Scopus (16) Google Scholar, 16Schneider B. Muller R. Frank R. Lingens F. Biol. Chem. Hoppe-Seyler. 1993; 374: 489-496Crossref PubMed Scopus (19) Google Scholar, 17Liu J.-Q. Kurihara T. Esaki N. Soda K. J. Biochem. 1994; 116: 248-249Crossref PubMed Scopus (13) Google Scholar, 18Kurihara T. Liu J.-Q. Nardi-Dei V. Koshikawa H. Esaki N. Soda K. J. Biochem. 1995; 117: 1317-1322Crossref PubMed Scopus (76) Google Scholar). His20 of L-DEX from Pseudomonas cepacia MBA4 was proposed to act as a catalytic base as shown in Fig. 1B(15Asmara W. Murdiyatmo U. Baines A.J. Bull A.T. Hardman D.J. Biochem. J. 1993; 292: 69-74Crossref PubMed Scopus (16) Google Scholar). However, replacement of His19 of L-DEX YL, corresponding to His20 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(17Liu J.-Q. Kurihara T. Esaki N. Soda K. J. Biochem. 1994; 116: 248-249Crossref PubMed Scopus (13) Google Scholar). 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 Asp10, Asp180, Lys151, Ser175, Arg41, Thr14, Tyr157, and Asn177(18Kurihara T. Liu J.-Q. Nardi-Dei V. Koshikawa H. Esaki N. Soda K. J. Biochem. 1995; 117: 1317-1322Crossref PubMed Scopus (76) Google Scholar). However, we could not show which mechanism of those shown in Fig. 1 is involved in the reaction and identify the catalytic residue.We conducted single and multiple turnover enzyme reactions in H218O 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 Asp10 acts as a catalytic nucleophile.EXPERIMENTAL PROCEDURESMaterialspBA5 encoding L-DEX YL was constructed as described in the previous paper(8Nardi-Dei V. Kurihara T. Okamura T. Liu J.-Q. Koshikawa H. Ozaki H. Terashima Y. Esaki N. Soda K. Appl. Environ. Microbiol. 1994; 60: 3375-3380Crossref PubMed Google Scholar). The 1.5-kilobase pair PstI-BamHI fragment of pBA5 containing the 3ʹ-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. H218O (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 EndopeptidaseLysyl 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 Leu11, Ser176, and Arg185 by Lys was carried out by the method of Kunkel(19Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4543) Google Scholar). The mutant enzymes and synthetic mutagenic primers are as follows (the underlines indicate the mutagenized nucleotides): L11K, 5ʹ-ACAGCGTACCGTACTTGTCGAAGGCAATACC-3ʹ; S176K, 5ʹ-GCGTTCTTCGACACGAACAGG-3ʹ; R185K, 5ʹ-GAAGCCGAAGTATTTCGCCCCCG-3ʹ. 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 EnzymeRecombinant 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-β-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 AssayL-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.(20Iwasaki I. Utsumi S. Hagino K. Ozawa T. Bull. Chem. Soc. Jpn. 1956; 29: 860-864Crossref Google Scholar). 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 H218OFor a typical single turnover experiment, 200 nmol of the wild-type L-DEX YL in 50 μl of 50 mM Tris-H2SO4 buffer (pH 9.5) was lyophilized. The reaction was initiated by dissolving the dried enzyme in 50 μl of H218O 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-H2SO4 (pH 9.0) previously lyophilized were mixed in 50 μl of H218O and incubated at 30°C for 24 h. The reaction mixtures were ultrafiltrated, diluted 10-fold with 50% acetonitrile/H2O (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 H2OWild-type L-DEX YL was labeled with 18O under the same conditions as the multiple turnover reaction in H218O 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-H2SO4 buffer (pH 9.5), and the recovered enzyme was used as the 18O-labeled enzyme to catalyze single and multiple turnover reactions in H2O according to the same procedures as described above.Digestion of 18O-Labeled L-DEX T15 with Lysyl Endopeptidase10 nmol of lyophilized L-DEX T15, 1 μmol of L-2-chloropropionate, and 2.5 μmol of Tris-H2SO4 (pH 9.0) were mixed in 50 μl of H218O 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 H218O without substrate was used as a control.LC/MS Analysis of the Proteolytic DigestThe 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 Asp10 and Asp180, the proteolytic digest of the enzyme incubated in H218O with or without a substrate was applied to a C18 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 SpectrometryThe MS/MS daughter ion spectra were obtained in the triple quadrupole daughter scan mode by selectively introducing the peptides containing Asp10 (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 × 1014 molecules/cm2.Amino Acid SequencingThe amino acid sequences of peptides were determined with a fully automated protein sequencer PPSQ-10 (Shimadzu, Kyoto, Japan).RESULTSSingle and Multiple Turnover Reactions of Wild-type L-DEX YL in H218OUnder the single turnover conditions, less than 10% D-lactate produced in H218O 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 H218O. 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Single and Multiple Turnover Reactions of 18O-Labeled L-DEX YL in H2OAfter the multiple turnover reaction in H218O, 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 H2O 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 H2O. The spectra were obtained under the same conditions as described in the Fig. 2 legend. A, single turnover reaction. B, multiple turnover reaction.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Construction, Purification, and Characterization of L-DEX T15To 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 FormedL-DEX T15 was used to carry out a multiple turnover reaction in H218O 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 Asp10.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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tabled 1 Open table in a new tab L-DEX T15 was incubated in H218O 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 Asp10 and Asp180, 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 H218O 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 Asp10 and Asp180. Panels A and B, Gly6-Lys11 hexapeptide derived from L-DEX T15 incubated in H218O with (B) or without (A) substrate. Panels C and D, Asn177-Lys185 nonapeptide derived from L-DEX T15 incubated in H218O with (D) or without (C) substrate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Analysis of the Active Site Peptide by Tandem MS/MS SpectrometryFragmentations 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 Yʹ 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 Asp10 of the enzyme during the dehalogenation reaction.Figure 6:Tandem MS/MS daughter ion spectra of 18O-labeled and unlabeled active site peptides (Gly6-Lys11). A, unlabeled peptide (m/z 650.2, the parent ion). B, 18O-labeled peptide (m/z 654.5, the parent ion).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONSingle and multiple turnover reaction studies conducted in H218O 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.Asp10 and Asp180 were shown to be important for catalysis by our site-directed mutagenesis experiments(18Kurihara T. Liu J.-Q. Nardi-Dei V. Koshikawa H. Esaki N. Soda K. J. Biochem. 1995; 117: 1317-1322Crossref PubMed Scopus (76) Google Scholar). 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 H218O. LC/MS and tandem MS/MS analysis showed that an oxygen atom of solvent water was incorporated into the carboxylate group of Asp10, but not into Asp180. Accordingly, the dehalogenation reaction of L-DEX probably proceeds through the ester intermediate mechanism in which Asp10 functions as a nucleophile (Fig. 1A). Since two 18O atoms were incorporated into Asp10, both oxygen atoms of the carboxylate group of Asp10 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 (21Koonin E.V. Tatusov R.L. J. Mol. Biol. 1994; 244: 125-132Crossref PubMed Scopus (266) Google Scholar). These proteins include different types of phosphatases and numerous uncharacterized proteins from eubacteria, eukaryotes, and Archaea. Among all of these proteins, Asp10 is completely conserved. This suggests the essential role of Asp10 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(22Lacourciere G.M. Armstrong R.N. J. Am. Chem. Soc. 1993; 115: 10466-10467Crossref Scopus (151) Google Scholar), haloalkane dehalogenase from Xanthobacter autotrophicus GJ10(23Pries F. Kingma J. Pentenga M. van Pouderoyen G. Jeronimus-Stratingh C.M. Bruins A.P. Janssen D.B. Biochemistry. 1994; 33: 1242-1247Crossref PubMed Scopus (98) Google Scholar), and (4-chlorobenzoyl)coenzyme A dehalogenase from Pseudomonas sp. strain CBS3(24Yang G. Liang P.-H. Dunaway-Mariano D. Biochemistry. 1994; 33: 8527-8531Crossref PubMed Scopus (51) Google Scholar). 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, Asp10 of L-DEX probably acts as a nucleophile to attack the α-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 Asp10 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) INTRODUCTIONL-2-Haloacid dehalogenase (L-DEX) ( 1The abbreviations used are: L-DEXL-2-haloacid dehalogenaseL-DEX YLthermostable L-2-haloacid dehalogenase from Pseudomonas sp. YLMSmass spectrometryLCliquid chromatography. )catalyzes the hydrolytic dehalogenation of L-2-haloacids with inversion of the C2 configuration producing the corresponding D-2-hydroxy acids. The enzymes have been isolated from various bacteria and characterized(1Goldman P. Milne G.W.A. Keister D.B. J. Biol. Chem. 1968; 243: 428-434Abstract Full Text PDF PubMed Google Scholar, 2Little M. Williams P.A. Eur. J. Biochem. 1971; 21: 99-109Crossref PubMed Scopus (51) Google Scholar, 3Motosugi K. Esaki N. Soda K. Agric. Biol. Chem. 1982; 46: 837-838Google Scholar, 4Tsang J.S.H. Sallis P.J. Bull A.T. Hardman D.J. Arch. Microbiol. 1988; 150: 441-446Crossref Scopus (52) Google Scholar, 5Jones D.H.A. Barth P.T. Byrom D. Thomas C.M. J. Gen. Microbiol. 1992; 138: 675-683Crossref PubMed Scopus (50) Google Scholar, 6Liu J.-Q. Kurihara T. Hasan A.K.M.Q. Nardi-Dei V. Koshikawa H. Esaki N. Soda K. Appl. Environ. Microbiol. 1994; 60: 2389-2393Crossref PubM" @default.
• W2008584836 created "2016-06-24" @default.
• W2008584836 creator A5000872539 @default.
• W2008584836 creator A5019049856 @default.
• W2008584836 creator A5034996400 @default.
• W2008584836 creator A5035751988 @default.
• W2008584836 creator A5038857193 @default.
• W2008584836 date "1995-08-01" @default.
• W2008584836 modified "2023-10-15" @default.
• W2008584836 title "Reaction Mechanism of L-2-Haloacid Dehalogenase of Pseudomonas sp. YL" @default.
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