University of Groningen Kinetic mechanism of the enantioselective conversion of styrene oxide by epoxide hydrolase from Agrobacterium radiobacter AD1 Rink,

Epoxide hydrolase from Agrobacterium radiobacter AD1 catalyzes the enantioselective hydrolysis of styrene oxide with an E value of 16. The (R)-enantiomer of styrene oxide is first converted with a kcat of 3.8 s-1, and the conversion of the (S)-enantiomer is inhibited. The latter is subsequently hydrolyzed with a kcat of 10.5 s-1. The pre-steady-state kinetic parameters were determined for both enantiomers with stopped-flow fluorescence and rapid-quench techniques. For (R)-styrene oxide a fourstep mechanism was needed to describe the data. It involved the formation of a Michaelis complex that is in rapid equilibrium with free enzyme and substrate, followed by rapid and reversible alkylation of the enzyme. A unimolecular isomerization of the alkylated enzyme precedes the hydrolysis of the covalent intermediate, which could be observed due to an enhancement of the intrinsic protein fluorescence during this step. The conversion of (S)-styrene oxide could be described by a three-step mechanism, which also involved reversible and rapid formation of an ester intermediate from a Michaelis complex and its subsequent slow hydrolysis as the rate-limiting step. The unimolecular isomerization step has not been observed for rat microsomal epoxide hydrolase, for which a kinetic mechanism was recently established [Tzeng, H.-F., Laughlin, L. T., Lin, S., and Armstrong, R. N. (1996) J. Am. Chem. Soc. 118, 94369437]. For both enantiomers of styrene oxide, the Km value was much lower than the substrate binding constant KS due to extensive accumulation of the covalent intermediate. The enantioselectivity was more pronounced in the alkylation rates than in the rate-limiting hydrolysis steps. The combined reaction schemes for (R)and (S)-styrene oxide gave an accurate description of the epoxide hydrolase catalyzed kinetic resolution of racemic styrene oxide. Epoxide hydrolases can convert epoxides to their corresponding diols by the addition of a water molecule without the use of a cofacter. About fifteen epoxide hydrolase genes have been isolated from different organisms, including mammals, insects, plants, fungi, and bacteria. All of the enzymes appear to be structurally and mechanistically similar, including two recently cloned bacterial epoxide hydrolases that are related to the ones cloned from higher organisms (1-4). The main incentive for studying these enzymes is their key role in the detoxification of xenobiotic compounds in the liver, but increasing attention is given to their great potential in biocatalysis. Epoxide hydrolases from eukaryotic and prokaryotic sources were found to be applicable for the kinetic resolution of racemic mixtures of epoxides, and high enantiomeric excesses have been reported (5-7). Most detailed studies have been performed on microsomal and soluble epoxide hydrolases from mammalian origin. These include substrate and inhibitor studies, sitedirected mutagenesis studies on catalytic residues, and a recently performed pre-steady-state kinetic analysis with rat microsomal epoxide hydrolase (8-10). Epoxide hydrolases are classified as R/â-hydrolase fold enzymes (2, 11). The topology of this class of enzymes shows two domains, a main domain that consists of a central â-sheet surrounded by R-helices with the catalytic residues excursing on top of the sheet at loops, and a cap domain that consists predominantly of R-helices and is involved in substrate binding. The mammalian epoxide hydrolases contain an additional N-terminal domain, which has sequence similarity to another class of hydrolytic enzymes in the case of the soluble epoxide hydrolase (12), and a membrane anchor involved in bile acid transport in the case of microsomal epoxide hydrolase (13). The bacterial epoxide hydrolases cloned up to now are simpler enzymes, since they do not contain these extra N-terminal domains (3, 4). However, they have not yet been studied in great detail. The epichlorohydrin epoxide hydrolase (EchA)1 that was used for this study was isolated from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole source of carbon and energy. The corresponding gene was overexpressed in Escherichia coli BL21(DE3) (4, 14). The epoxide hydrolase is a 34 kDa monomeric enzyme that has a broad substrate range. On the basis of the sequence analysis and the behavior of site* To whom correspondence should be addressed: Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands. Tel: 31-50-3634209. Fax: 31-503634165. E-mail: [email protected]. 1 Abbreviation: EchA, epichlorohydrin epoxide hydrolase from Agrobacterium radiobacter AD1. 18119 Biochemistry 1998, 37, 18119-18127 10.1021/bi9817257 CCC: $15.00 © 1998 American Chemical Society Published on Web 12/02/1998 specific mutants, EchA was also classified as an R/âhydrolase fold enzyme. It has a reaction mechanism in which Asp107 performs a nucleophilic attack on the primary carbon atom of the epoxide ring, leading to a covalently bound ester intermediate (Figure 1). The Asp246-His275 pair, supported by Asp131, then activates a water molecule that hydrolyzes the ester bond at the carbonyl function of Asp107, and product is released (4). It turned out that EchA could discriminate between the enantiomers of styrene oxide and substituted variants thereof, affording the production of enantiomerically pure epoxides (7). Styrene oxide showed peculiar kinetics of conversion, since the (R)-enantiomer was completely hydrolyzed before the (S)-enantiomer, but the latter was subsequently converted at a much higher rate. How this sequential hydrolysis can be explained by reaction rates at the enzyme active site is the focus of this study. To this end, we investigated the kinetics of styrene oxide conversion by means of steady-state and pre-steady-state kinetic experiments. Stopped-flow fluorescence and rapidquench techniques were used to resolve the kinetic constants. The resulting kinetic constants could quantitatively describe the enantioselective conversion of racemic styrene oxide. MATERIALS AND METHODS Materials. (R)and (S)-styrene oxide and (R)and (S)-1phenyl-1,2-ethanediol were purchased from Aldrich and were 97% enantiomerically pure as was confirmed by gas chromatographic analysis. Protein Expression and Purification. E. coli BL21(DE3) with plasmid pEH20 containing the epichlorohydrin epoxide hydrolase gene echA of A. radiobacter AD1 under control of a T7 promoter was used for the expression of epoxide hydrolase. Protein was expressed and purified as described before (4). Enzyme was kept at 4 °C in PEMAG buffer (10 mM phosphate, pH 7.5, 1 mM EDTA, 1 mM â-mercaptoethanol, 0.02% sodium azide, and 10% (v/v) glycerol) and could be stored for over six months without losing activity. Stopped-Flow Fluorescence. Stopped-flow fluorescence experiments were performed at 30 °C on an Applied Photophysics model SX.17MV apparatus. The tryptophan residues were excited at a wavelength of 290 nm, and the resulting fluorescence cut-off emission was recorded after passage through a 305 nm cutoff filter. Stock solutions of (R)and (S)-styrene oxide were prepared in acetonitrile and were freshly diluted in TEMA buffer (50 mM Tris-SO4, pH 9.0, 1 mM EDTA, 1 mM â-mercaptoethanol, and 0.02% sodium azide) to a final concentration of 0.5% (v/v) acetonitrile. Low concentrations of acetonitrile did not affect enzyme activity. Enzyme solutions were also prepared in TEMA buffer, and all mentioned concentrations are those after mixing. For single-turnover reactions the enzyme was concentrated in TEMA buffer by using an Amicon ultrafiltration cell with a PM-10 filter. Rapid-Quench Experiments. Single-turnover experiments were performed at 30 °C on a RQF-63 rapid-quench-flow apparatus from Kintek Instruments. Concentrated enzyme and a freshly prepared styrene oxide solution from an acetonitrile stock were mixed, and the total volume of 100 μL was quenched with KOH to a final concentration of 0.7 M. The instability of styrene oxide under strongly basic conditions was overcome by directly injecting the quenched reaction mixture (217 μL) into a tube containing 61 μL of 2 M HCl, 200 μL of 1 M borate buffer, pH 9.0, and 0.5 mL of diethyl ether with 10 μM 1,9-dichlorononane as the internal standard. These quenching conditions could not prevent that a small fraction of the covalent ester intermediate was still hydrolyzed, but increasing the concentration of KOH or the use of HCl as a quenching agent resulted in nonenzymatic hydrolysis of styrene oxide. Quantitative analysis of the styrene oxide in the ether phase was done by gas chromatography. The 1-phenyl-1,2ethanediol that remained in the water phase was derivatized with 2,2-dimethoxypropane to give a hemiacetal that was easily detected by gas chromatography (15). For this procedure, 400 μL of the waterphase was saturated with sodium chloride and the diol was extracted with 0.6 mL of 2,2-dimethoxypropane with 10 μM 1,9-dichlorononane as the internal standard. The organic phase was then transferred to a sealed tube with 0.2 g of Amberlite IR-120 (H+), a strongly acidic cation exchanger that is coated on polystyrene, and the derivatization reaction was completed by vigorous shaking at 30 °C for 1 h. Both the styrene oxide and the hemiacetal derivative of 1-phenyl-1,2-ethanediol were analyzed by gas chromatography using a 0.2 mm × 25 m CP-Wax-57CB column (Chrompack) and a flameionization detector. Steady-State Parameters. The kcat values of (R)and (S)styrene oxide were determined by following substrate depletion by gas chromatography. A vial with a sufficient amount of enzyme and 5 mM substrate was incubated at 30 °C, and at different times within 15 min samples of 1 mL were taken. The sample was extracted with 2 mL of diethyl ether with 10 μM 1,9-dichlorononane as the internal standard. The organic phase was analyzed on a 0.2 mm × 25 m CP-Wax57CB column using a gas chromatograph equipped with a flame-ionization detector. FIGURE 1: Proposed reaction mechanism for epoxide hydrolase. (A) Nucleophilic attack of Asp107 at the least-hindered carbon atom of styrene oxide, leading to the formation of a covalent ester intermediate. Presumably, an unidentified proton donor (B-H) assists in ring opening. (B) Proton abstraction from a water molecule by the His275-Asp246 pair, assisted by Asp131, and subsequent hydrolysis of the intermediate. 18120 Biochemistry, Vol. 37, No. 51, 1998 Rink and Janssen