Substrate-dependent hysteretic behavior in StEH1-catalyzed hydrolysis of styrene oxide derivatives Diana Lindberg, Adolf Gogoll and Mikael Widersten Department of Biochemistry and Organic Chemistry, Uppsala University, Sweden Soluble epoxide hydrolases (EC 3.3.2.10) make up a large group of enzymes catalyzing the hydrolysis of alkyl and aryl epoxides into the corresponding vicinal diols [1,2]. They fulfill various functions in host organisms, including detoxification by hydrolysis of endogenous and exogenous epoxides, regulation of cell signaling by hydrolysis of epoxide-containing bioactive lipids, or participation in the secondary metabolism of microorganisms. In plants, epoxide hydrolases have been suggested to contribute to path- ogen defense systems through hydrolysis of epoxy- containing hydroxyl fatty acids. The diol products from the latter reaction show antifungal activity [3] and are established substrates for several plant iso- enzymes as precursors in cutin synthesis [4]. The inde- pendence from cofactors in combination with, in some cases, high catalytic efficiencies and enantio- selectivities has created an interest in using epoxide hydrolases as biocatalysts in the production of fine chemicals [5,6]. Styrene oxide (SO) and derivatives thereof are important molecules as chiral and prochiral precursors in asymmetric synthesis. These compounds are also relevant for their toxicological impact [7]. Solanum tuberosum epoxide hydrolase 1 (StEH1) has previously been investigated using different SO derivatives as Keywords epoxide hydrolase; kinetic mechanism; pre- steady state; regiospecificity; styrene oxide Correspondence M. Widersten, Box 576, SE-751 23 Uppsala, Sweden Fax: +46 0 18 55 8431 Tel: +46 0 18 471 4992 E-mail: mikael.widersten@biorg.uu.se Website: http://www.biorg.uu.se (Received 12 September 2008, revised 20 October 2008, accepted 22 October 2008) doi:10.1111/j.1742-4658.2008.06754.x The substrate selectivity and enantioselectivity of Solanum tuberosum epoxide hydrolase 1 (StEH1) have been explored by steady-state and pre- steady-state measurements on a series of styrene oxide derivatives. A prefer- ence for the (S)- or (S,S)-enantiomers of styrene oxide, 2-methylstyrene oxide and trans-stilbene oxide was established, with E-values of 43, 160 and 2.9, respectively. Monitoring of the pre-steady-state phase of the reaction with (S,S)-2-methylstyrene oxide revealed two observed rates for alkylenzyme for- mation. The slower of these rates showed a negative substrate concentration dependence, as did the rate of alkylenzyme formation in the reaction with the (R,R)-enantiomer. Such kinetic behavior is indicative of an additional, off-pathway step in the mechanism, referred to as hysteresis. On the basis of these data, a kinetic mechanism that explains the kinetic behavior with all tested substrates transformed by this enzyme is proposed. Regioselectivity of StEH1 in the catalyzed hydrolysis of 2-methylstyrene oxide was determined by 13 C-NMR spectroscopy of 18 O-labeled diol products. The (S,S)-enantiomer is attacked exclusively at the C-1 epoxide carbon, whereas the (R,R)-enantiomer is attacked at either position at a ratio of 65 : 35 in favor of the C-1 carbon. On the basis of the results, we conclude that differ- ences in efficiency in stabilization of the alkylenzyme intermediates by StEH1 are important for enantioselectivity with styrene oxide or trans-stilbene oxide as substrate. With 2-methylstyrene oxide, slow conformational changes in the enzyme also influence the catalytic efficiency. Abbreviations 2-MeSO, 2-methylstyrene oxide; ES, enzyme–substrate; SO, styrene oxide; StEH1, Solanum tuberosum epoxide hydrolase 1; TSO, trans-stilbene oxide. FEBS Journal 275 (2008) 6309–6320 ª 2008 The Authors Journal compilation ª 2008 FEBS 6309 substrates, both in buffer and immobilized on different matrices [8–15]. As with other isoenzymes of the a ⁄ b-hydrolase fold family, the kinetic mechanism of StEH1 catalysis in its simplest form consists of the three steps outlined in Fig. 1. Formation of the Michaelis complex is fol- lowed by a nucleophilic attack by the active site Asp105 carboxylate to generate a covalent alkyl- enzyme intermediate [16,17]. The alkylation half-reac- tion is also dependent on electrophilic catalysis by two active-site Tyr residues (Tyr154 and Tyr235) [13,18,19]. Catalytic turnover is subsequently finalized by a general base-assisted hydrolysis of the alkylen- zyme, facilitated by the imidazole of His300, and product release [10,20–22]. In principle, all of the described steps may influence the overall stereospeci- ficity of catalysis. The active site architecture, including the spatial arrangement of catalytic groups, is expected to place restrictions on the productive substrate-binding modes. For instance, good alignments of the epoxide for nucleophilic attack on an electrophilic carbon by the carboxylate and for the Tyr phenols previously mentioned are crucial for effective catalysis. Alkyl- enzyme formation depends on efficient activation of the nucleophile via deprotonation of the carboxyl group, stabilization of the leaving group oxide through hydrogen bonding to the Tyr pair, and subse- quent protonation of the alkylenzyme anion formed [23]. In the final catalytic step, efficient hydrolysis requires that the alkylenzyme, following attack by water, undergoes a structural change in order to stabi- lize the formed oxyanion through amide backbone interactions [24–26]. Enzyme structure flexibility is therefore also expected to influence catalytic efficiency and enantiopreference. StEH1 exhibits reasonable stereoselectivity with SO, with different regiospecificity in the initial nucleophilic attack by the enzyme on the different enantiomers, resulting in an enantioconvergent synthesis of (R)-1- phenylethane-1,2-diol [9]. Other studies on the hydroly- sis of trans-stilbene oxide (TSO) suggest that the kinetic mechanisms, under certain conditions, may differ with substrate enantiomers [15]. Stereodiscrimination with glycidyl-4-nitrobenzoate displayed by the rat microsomal epoxide hydrolase has been attributed primarily to the hydrolysis half-reac- tion [20], whereas it is the alkylation step that has been shown to mainly influence the enantiospecificity in SO hydrolysis by the Agrobacterium radiobacter AD1 enzyme [27]. This illustrates that the mechanisms caus- ing stereospecificity may vary with isoenzyme and⁄ or substrate. In addition to the enzyme-afforded catalytic steps, a further level of reaction complexity is added with SO derivatives as substrates. The phenyl substituent of these compounds can assist in stabilizing electron-rich reaction intermediates, hence affecting the regioselec- tivity in the ring-opening half-reaction by directing the attack away from the otherwise more reactive, least substituted, of the two oxirane carbons [28,29]. In this work, we aimed to systematically analyze the causes of StEH1 substrate selectivity, using different SO structural analogs. SO, 2-methyl styrene oxide (2-MeSO) and TSO, with increasing size and hydro- phobicity on the C-2 substituent, were used to derive these structure–function relationships. In addition, the regioselectivity in StEH1-catalyzed hydrolysis of 2-MeSO was analyzed by 13 C-NMR spectroscopy of 18 O-labeled diol products. Results and Discussion Steady-state measurements The ratio of k cat ⁄ K m values for two substrates is a direct measure of the enzymatic substrate discrimina- tion. The lower K m values obtained as a result of these measurements with the (S)- or ( S ,S )-enantiomers as compared to their (R)or(R,R) counterparts results in relatively high enantiospecificity values [E = (k cat S ⁄ K m S ) ⁄ (k cat R ⁄ K M R )] for SO and 2-MeSO; 43-fold and 160-fold, respectively (Table 1). This degree of enantiospecificity in favor of (S)-SO is in agreement with previous studies, in which E-values have been estimated from specific enzyme activities and product composition analyses following hydrolysis of racemic SO [9,12]. When comparing the steady-state parame- ters derived from the reactions with (S,S)-2-MeSO and Fig. 1. Kinetic mechanism of StEH1-catalyzed epoxide hydrolysis. The mechanism includes the formation of a covalent alkylenzyme, formed with rate k 2 , following the Michaelis complex formation. The alkylenzyme is subsequently hydrolyzed, with rate k 3 ,to restore the enzyme. K S , the equilibrium dissociation constant of the ES complex, is the ratio of the dissociation and association rate constants, k )1 ⁄ k 1 . Enzyme-catalyzed styrene oxide hydrolysis D. Lindberg et al. 6310 FEBS Journal 275 (2008) 6309–6320 ª 2008 The Authors Journal compilation ª 2008 FEBS the corresponding SO enantiomer, it is clear that (S,S)-2-MeSO is the favored substrate. The k cat (S,S)-2- MeSO is the highest recorded for this enzyme with any substrate, and is approximately 10-fold higher than k cat for the (R,R)-enantiomer (Table 1). The enantiomer discrimination displayed by the enzyme in SO or 2-MeSO hydrolysis is virtually lost with TSO. The enzyme exhibits comparably low turn- over rates with TSO, but low K m values compensate to yield high values of k cat ⁄ K m (Table 1). Hence, energy gained from favorable enzyme–substrate (ES) interac- tions is more important than turnover rate in deciding catalytic efficiency. A modeling study [26] has suggested that the size and shape of the active site is well adapted for fitting the TSO enantiomers, restricting binding to snugly fitted conformations for both enantiomers. The resulting free energy realized in the ES complexes can be calculated from the K S parameters (Table 2) to be )25 and )28 kJÆmol )1 for the (R,R)- and ( S ,S)-enantiomers, respectively. The corresponding energies for the SO or (S,S)-2-MeSO substrates are > 6 kJÆmol )1 less favor- able in those Michaelis complexes. This ground state stabilization of the TSO enantiomers will be unfavor- able for subsequent chemical steps, by increasing the energy barrier for formation of alkylenzyme, reflected as comparably lower values of k 2 (Table 2). In the (S)-enantiomer series, where data are available for all tested derivatives, the alkylation rate for TSO is the slowest by a factor of at least three-fold. The snug fit of the TSO enantiomers in the active site may further act to prevent the dynamic movement necessary for effi- cient hydrolysis of the alkylenzyme, which is possibly reflected in the relatively low values of the k 3 and k cat parameters (Tables 1 and 2). The kinetic mechanism involved in StEH1 hydrolysis of TSO and SO It is presupposed within the simple kinetic scheme out- lined in Fig. 1 that a rapid equilibrium forms between free enzyme, substrate, and the Michaelis complexes. Another assumption made within the model is that product release is not rate-limiting for turnover, as supported by the poor inhibition of the hydrolysis products in these reactions [9,10]. From the kinetic model in Fig. 1, it can be seen that the corresponding steady-state rate law is described by Eqn (1), where k cat is composed of the first factor in the numerator and K m of the first term in the denominator: v ¼ k 2 k 3 ðk 2 þ k À2 þ k 3 Þ ½E 0 ½S K S ðk À2 þ k 3 Þ ðk 2 þ k À2 þ k 3 Þ þ½S ð1Þ In the pre-steady-state measurements, formation of the alkylenzyme reaction intermediates was recorded as concomitant decreases in intrinsic Trp fluorescence [13]. The fluorescence decrease under pseudo-first-order conditions of both enantiomers of SO and TSO, as Table 1. Steady-state kinetic parameters and E-values of the StEH1-catalyzed hydrolysis of styrene oxide and 2-substituted styrene oxide derivatives. Substrate k cat (s )1 ) K m (lM) k cat ⁄ K m (s )1 ÆlM –1 ) E (S) ⁄ (R) (S)-SO 10 ± 1.5 220 ± 88 0.047 ± 0.0012 43 (R)-SO > 9 > 400 0.0011 ± 0.000096 (S,S)-2-MeSO 52 ± 2.6 99 ± 1.7 0.53 ± 0.062 160 (R,R)-2-MeSO 5.7 ± 1.5 1400 ± 590 0.0034 ± 0.0018 (S,S)-TSO 0.91 ± 0.063 0.31 ± 0.0091 2.9 ± 0.72 2.9 (R,R)-TSO 15 ± 1.1 16 ± 2.7 1.0 ± 0.11 Table 2. Pre-steady-state kinetic and thermodynamic parameters of StEH1-catalyzed hydrolysis of styrene oxide derivatives. Substrate K S (lM) k 0 + k )0 (s )1 ) k 2 (s )1 ) k )2 (s )1 ) k 3 (s )1 ) k 5 + k )5 (s )1 ) k )2 + k 3 (s )1 ) k 2 ⁄ K S (s )1 ÆlM )1 ) (S)-SO > 400 – > 85 – – – 55 ± 2.9 0.11 ± 0.0061 (R)-SO > 600 – > 16 – – – 41 ± 2.2 0.013 ± 0.0031 (S,S)-2-MeSO > 500 80 > 300 – – 20 310 ± 12 0.30 ± 0.036 (R,R)-2-MeSO – 80 – – – 10 – – (S,S)-TSO 14 ± 7.7 – 29 ± 5.8 14 ± 1.9 1.4 ± 0.36 – – 3.3 ± 1.5 (R,R)-TSO 42 ± 37 – 51 ± 6.9 26 ± 9.4 32 ± 0.37 – – 2.6 ± 2.0 D. Lindberg et al. Enzyme-catalyzed styrene oxide hydrolysis FEBS Journal 275 (2008) 6309–6320 ª 2008 The Authors Journal compilation ª 2008 FEBS 6311 well as the (R,R)-enantiomer of 2-MeSO, follows first- order exponential decays, allowing for extraction of observed rates (k obs ) for the reactions from the ES complex to alkylenzyme. Observed rates for the reac- tions with either enantiomer of TSO show hyperbolic substrate dependence (Fig. 2A). Fitting of Eqn (2) to data was used to determine the equilibrium dissocia- tion constants of the ES complex (K S ), as well as the rate constants for formation and decomposition of the alkylenzyme: k 2 and k )2 + k 3 , respectively. The values of k –2 and k 3 were extracted from the expression for k cat (Table 2): k obs ¼ k 2 ½S K S þ½S þðk À2 þ k 3 Þð2Þ The obtained rate constants and K S values for the reaction with (S,S)-TSO agree with previous studies on the pH dependence of StEH1, which predicted an increase in k 2 with a concomitant decrease in k 3 when the pH is changed from 6.8 to 8.0 [13]. The resulting increase in accumulation of the alkylenzyme contrib- utes to lower the value of K m (S,S)-TSO from 5 lm at pH 6.8 to 0.31 lm at pH 8.0. As K S (S,S)-TSO is not significantly affected by the pH shift (Table 3), it A B C D Fig. 2. Observed rates, k obs , versus substrate concentration for tested SO derivatives. (A) (S,S)-TSO (unfilled squares) and (R,R)-TSO (filled squares). Solid lines represent the fits to Eqn (2). (B) (S)-SO (unfilled triangles) and (R)-SO (filled triangles). Solid lines represent the fits to Eqn (3). (C) Substrate dependence on observed higher rate, k obs1 , in the (S,S)-2-MeSO reaction (unfilled circles). The solid line represents the fit to Eqn (3). Inset: average of five traces of fluorescence decay in the presence of 1000 l M (S,S)-2-MeSO and 2 lM StEH1. Lines repre- sent a single (dashed line) or a double (straight line) exponential fit to the experimental data. Residual plots of the respective fits are shown in boxes. (D) Substrate dependence of the low rate, k obs2 , with (S,S)-2-MeSO (half-filled circles) and (R,R)-2-MeSO (filled circles). Solid lines represent the fits to Eqn (4). See Table 2 for determined values of kinetic parameters. Enzyme-catalyzed styrene oxide hydrolysis D. Lindberg et al. 6312 FEBS Journal 275 (2008) 6309–6320 ª 2008 The Authors Journal compilation ª 2008 FEBS follows that the changes in formation and decomposi- tion rates of the alkylenzyme are mainly responsible for the lowering of the K m value. The lower K m (S,S)-TSO in turn increases the k cat ⁄ K m (S,S)-TSO value. With (R,R)-TSO, however, the increase in pH from 6.8 to 8.0 causes a decrease in k 2 (Table 3), which affects both k cat and K m adversely to yield a lower value of k cat ⁄ K m (R,R)-TSO at the higher pH. The combined effect on the hydrolysis of the TSO enantiomers is a shift in enantiopreference from three-fold in favor of ( R ,R)- TSO at pH 6.8 [15] to a three-fold preference for the (S,S)-enantiomer at pH 8.0. If K S >> [S], the hyperbolic concentration depen- dence given by Eqn (2) simplifies to a linear relation- ship (Eqn 3). In such cases, as observed in the reactions with the SO enantiomers (Fig. 2B), the enzyme is far from saturating substrate concentrations, and direct determination of the individual parameters is impossible. k obs ¼ k 2 K S ½Sþðk À2 þ k 3 Þð3Þ In spite of the fact that values of k 2 could not be determined, we propose that in the reactions with SO, the main contributing factor to the observed enantio- specificity arises from the different rates of alkyl- enzyme formation. It can be deduced from Eqn (1) that the value of K m is dependent on the relationship between k 2 and K S. Hence, if K S reaches high relative values (i.e. low ES stabilization), a relatively higher degree of alkylenzyme accumulation is required [k 2 >(k –2 + k 3 )] in order to account for the deter- mined value of K m (S)-SO of 220 lm , which is consider- ably lower than the K S (S)-SO (> 400 lm). With (R)-SO, the pre-steady-state measurements show that the increase in observed rates with substrate concentration is comparably modest (Fig. 2B). This does not rule out the possibility that the alkylation rate may be similar to that of (S)-SO, but it would at the same time imply that K S (R)-SO is substantially higher for this enantio- mer. The enzyme active site provides a hydrophobic pocket with few opportunities for specific enthalpy- contributing interactions with these substrates other than possibly hydrogen bonding between the oxirane oxygen and the active site Tyr pair [15,26]. It therefore appears more likely that ES formation is an entropy- driven desolvation process whereby increased hydrophobicity in the substrate promotes ES forma- tion, lowering the value of K S . This agrees with the log P-values of the tested substrates (TSO, 3.4; 2-MeSO, 1.7; and SO, 1.6), whereby the most hydro- phobic derivative (TSO) also displays the lowest K S (Table 2). Following this assumption, the K S values of the different SO enantiomers are expected to be com- parable, and the relatively higher value of K m (R)-SO would mainly be an effect of a lower alkylation rate. As the ordinates of k obs at [S] = 0 provide measures of (k –2 + k 3 ), the ratio of the slopes of the fitted lines (k 2 ⁄ K S ) gives estimates of differences in alkylation rates. The results suggest that StEH1 catalyzes the formation of the alkylenzyme at an approximately 10-fold lower rate with the (R)-enantiomer (Table 2). Hence, in the reactions with SO, the main contributing factor to the observed enantiospecificity arises from the different rates of alkylenzyme formation. 2-MeSO pre-steady-state measurements suggest the presence of a hysteretic kinetic mechanism In order to fit the progression curves of transient Trp fluorescence quenching during the reaction with (S,S)- 2-MeSO, a double-exponential function had to be applied (inset in Fig. 2C). This results in two observed rates with individual amplitudes, suggesting the pres- ence of two alkylenzyme species. Plotting of the sub- strate dependence of k obs1,2 reveals that the faster observed rate increases linearly with increasing sub- strate concentration (Fig. 2C), similar to the (S)-SO case. The slower observed rate displays negative sub- strate dependence; k obs2 decreases with increasing sub- strate concentration (Fig. 2D). The presence of two observed rates may be explained by two different models: (a) an additional on-pathway alkylenzyme species that is formed with a distinct rate and displays distinct amplitude – this case has been demonstrated with (R)-SO in the reaction catalyzed by the epoxide hydrolase from A. radiobacter AD1 [27]; or (b) a mechanism involving a conformational change in the free enzyme, referred to as hysteresis [30,31], with alky- lenzyme species being formed within separated time frames. These intermediate species may be structurally Table 3. pH dependence of kinetic parameters of TSO hydrolysis. Parameter Substrates (S,S)-TSO (R,R)-TSO pH pH 6.8 8.0 6.8 8.0 k 2 (s )1 )18±2 a 29 ± 5.8 260 ± 56 b 51 ± 6.9 k 3 (s )1 ) 3.2 ± 0.06 a 1.4 ± 0.36 24 ± 3 a 32 ± 0.37 K S (lM)11±6 a 14 ± 7.7 36 ± 22 b 42 ± 37 a Data from [13]. b Data from [15]. D. Lindberg et al. Enzyme-catalyzed styrene oxide hydrolysis FEBS Journal 275 (2008) 6309–6320 ª 2008 The Authors Journal compilation ª 2008 FEBS 6313 identical but kinetically separated due to alternative pathways for formation. We have judged the linear model to be unlikely for two reasons. First, the substrate dependence of k obs2 displays a decrease with increasing concentration of (S,S)-2-MeSO, a behavior that requires a rate-limiting off-pathway conformational transition prior to sub- strate binding [32]. An on-pathway mechanism would result in either a substrate-dependent increase in observed rates, or in no such dependence. Second, as the mechanism described for the bacterial enzyme fol- lows a linear reaction pathway, the value of k obs2 must be ‡ k cat . In the present case, the limiting value of k obs2 is approximately 20 s )1 , which is considerably lower than the value of k cat (52 s )1 ). Our presently favored model therefore describes a mechanism similar to previously reported cases with other enzymes [33,34]. The model invokes two addi- tional reaction steps: (a) between two conformational states of the free enzyme, E … E¢; and (b) between the corresponding Michaelis complexes, ES … E¢S (Fig. 3A). With (S,S)-2-MeSO, it is assumed that only ES is capable of productive formation of alkylenzyme (k 6 > k 2 in Fig. 3D). The higher observed rate (k obs1 ) reflects the Michaelis mechanism E ‰ ES ‰ E-alky- l 1 fi E + diol 1 , displaying a substrate dependence according to Eqn (3), whereas the lower rate (k obs2 ) reflects the rate of alkylenzyme formation via a pathway involving a rate-limiting conformational change from E to E¢:Efi E¢ ‰ E¢S fi ES fi E-alkyl 1 fi E+ diol 1 . With this kinetic model, k obs2 can be expressed by Eqn (4) [33,34]. At low substrate concentrations, k obs2 tends towards k 0 + k –0 , and at higher substrate concen- trations towards k 5 + k –5 . Hence, if the transition E¢S ‰ ES occurs at a lower rate than in the free enzyme (E¢ ‰ E), i.e. substrate binding stabilizes one con- former, the observed rate will decrease with increased substrate concentration. k obs ¼ k 0 þðk 5 =K S Þ½S 1 þð½S=K S Þ þ k À0 þðk À5 =K 0 S Þ½S 1 þð½S=K 0 S Þ ð4Þ Kinetic cooperativity in hysteretic enzymes is shown by characteristic deviations from Michaelis–Menten kinetics: a burst or lag period in reaction progression curves and sigmoidal substrate saturation curves [31]. Owing to restrictions in the HPLC-based assay, it is not possible to resolve a lag period within the present data, which in the 2-MeSO reactions would be on a millisecond scale. Also, the data do not allow for a sig- nificant distinction between cooperative or non-cooper- ative substrate saturation. The steady-state parameters given in Table 1 are therefore determined after fitting the simplest (Michaelis–Menten) model. In the reactions with (R,R)-2-MeSO, the observed rates of transient fluorescence quenching follow single exponential decays, analogous to the reactions with SO or TSO. The substrate dependence of k obs , how- ever, displays a decrease with increasing substrate concentration, similar to the slow observed rate in the (S,S)-2-MeSO reaction (Fig. 2D). Hydrolysis of either 2-MeSO enantiomer therefore appears to be dependent D A B C Fig. 3. (A) Proposed kinetic mechanism of StEH1 hydrolysis of (S,S)-2-MeSO. The mechanism includes a fast, non-rate-limiting route to for- mation of the alkylenzyme followed by a rate-limiting hydrolysis step. A slow conformational change [E fi E¢ in (D)] is also included, describing the negative substrate dependence of k obs observed in the pre-steady-state measurements. (B) Discarded kinetic mechanism of (R,R)-2-MeSO hydrolysis, lacking a reaction route to explain the formation of two different product enantiomers. (C) Proposed kinetic mecha- nism of (R,R)-2-MeSO hydrolysis, including a rate-limiting conformational change of the free enzyme [E fi E¢ in (D)] and the formation of two distinct alkylenzymes [E-alkyl 1 and E¢-alkyl 2 in (D)]. (D) Proposed kinetic mechanism of StEH1-catalyzed epoxide hydrolysis of 2-MeSO. The model includes transitions between distinct forms of the free enzyme (E ‰ E¢) and Michaelis complexes (E¢S ‰ ES). In the reaction with (S,S)-2-MeSO, it is assumed that only the ES form is productive in forming the alkylenzyme, whereas with (R,R)-2-MeSO, both of the Michaelis complexes may form distinct alkylenzymes, resulting in a mixture of diol products. Simplifications of this model are also applicable to the catalyzed hydrolyses of SO and TSO enantiomers. Enzyme-catalyzed styrene oxide hydrolysis D. Lindberg et al. 6314 FEBS Journal 275 (2008) 6309–6320 ª 2008 The Authors Journal compilation ª 2008 FEBS on similar kinetic mechanisms. The presence of only one observed rate points towards a simpler model (Fig. 3B), where k obs = k 0 +[(k )0 K S ) ⁄ (K S + [S])] [35]. This model cannot, however, explain the forma- tion of the two different diol products (see discussion below), which requires the formation of two distinct alkylenzymes. Therefore, the model in Fig. 3C has been applied for fitting to data obtained in the (R,R)- 2-MeSO reaction. The resulting reaction pathways are E fi E¢ ‰ E¢S ‰ E¢-alkyl 2 and E fi E¢ ‰ E¢S ‰ ES ‰ E-alkyl 1 . The limiting value of k obs at high sub- strate concentrations is approximately two-fold higher than k cat (R,R)-2-MeSO (k 5 + k )5 in Table 2), which sup- ports the notion that the conformational change occur- ring prior to substrate binding is rate-limiting in this reaction. The physical differences between E and E¢ or ES and E¢S are at this stage highly speculative, and there is no evidence for conformational changes in the free enzyme from structural studies. There is the possibility, however, that distinct enzyme forms are required for productive interactions with substrates in different con- figurations in the respective active sites. It is clear that the structure of StEH1 provides ample space to allow 2-MeSO to bind in different modes within the active site, resulting in different ES conformers. One fact sup- porting this assumption is that both enantiomers of TSO, a 1,2-bisphenyl substituted epoxide, are readily accommodated in productive binding modes within the active site. Regiospecificity The basis for the observed regiospecificity in epoxide ring opening is difficult to rationalize even from ambi- tious structure–reactivity studies [28,29]. The carbon subjected to nucleophilic attack will be influenced by distinct intrinsic reactivities of the oxirane carbons as dictated by the substituents. Whereas bulky substitu- ents generally direct the nucleophile to attack the less hindered carbon, phenyl substituents may provide favorable electrostatic properties that overcome unfa- vorable steric features. In an enzyme-catalyzed reac- tion, the spatial alignment of catalytic groups, primarily the nucleophilic carboxylate and the electro- philic Tyr pair, may further affect catalytic rates to a degree that overcomes intrinsic reactivities. Monterde et al. have established the regiospecificity of the StEH1-catalyzed hydrolysis of SO [9]. Their results show that the carbon that is subject to nucleo- philic attack by the enzyme Asp is highly dependent on the enantiomer. The (S)-enantiomer was 98% attacked at the benzylic C-1, whereas (R)-SO reacted at the less substituted C-2 (93%). The detailed reasons for this clear shift are as yet unknown, but the result implicates different substrate-binding modes in the active site. Our kinetic studies indicate that alkylen- zyme formation is more efficient with the (S)-enantio- mer of SO by a factor of approximately 10, leading to a higher value of k cat ⁄ K m . The higher reaction rate may be due to more efficient activation of the epoxide oxygen through Lewis acid catalysis via more favor- able positioning of this enantiomer within the active site by the Tyr phenols and⁄ or more efficient stabili- zation of the transition state. The fact that C-2 is pri- marily attacked in the (R)-SO reaction may likewise be attributed to a binding mode restricting attack on C-1 for steric reasons and ⁄ or a lowering of the catalytic effect by the enzyme by placing the oxirane oxygen out of reach of the Tyr phenols. In this work, we have analyzed the regiospecificity during hydrolysis of the 2-MeSO enantiomers by 13 C-NMR spectroscopy via incorporation of an 18 O-labeled hydroxyl group at the attacked carbon in the presence of H 2 18 O. The results are partly in agree- ment with those observed in the SO reactions: (S,S)-2- MeSO is exclusively attacked at the benzylic carbon, with undetectable levels of incorporation of 18 O at C-2 (Table 4, Fig. 4). The regiospecificity in the reaction with (R,R)-2-MeSO produces a mixture of diol prod- ucts (Table 4), with 65% (S,R)-diol being formed as a result of attack at C-1, and the remaining 35% result- ing from attack at C-2, yielding the (R,S)-diol. Hence, the same degree of enantioconvergence present in the reaction with SO is not observed with 2-MeSO under the assay conditions used. Earlier studies with an epox- ide hydrolase-containing extract from Aspergillus terreus demonstrated product ratios after 2-MeSO hydrolysis similar to those observed in this study [36]. Other reports on the regioselectivity in the ring open- ing of 2-MeSO catalyzed by different epoxide hydro- Table 4. Integration of signals in 13 C-NMR spectra of 1-phenylpro- pane-1,2-diol. ND, not detectable. Substrate Diol carbon d Integral Ratio Sum (S,S)-2-MeSO CH 3 17.27 40.67 1.00 1.00 C-2- 18 O ND – – 1.02 C-2- 16 O 71.28 41.35 1.02 C-1- 18 O 77.48 20.59 0.51 1.02 C-1- 16 O 77.51 20.86 0.51 (R,R)-2-MeSO CH 3 17.30 17.66 1.00 1.00 C-2- 18 O 71.25 3.55 0.20 0.99 C-2- 16 O 71.28 13.89 0.79 C-1- 18 O 77.50 5.09 0.29 0.96 C-1- 16 O 77.52 11.82 0.67 D. Lindberg et al. Enzyme-catalyzed styrene oxide hydrolysis FEBS Journal 275 (2008) 6309–6320 ª 2008 The Authors Journal compilation ª 2008 FEBS 6315 lases have shown a modest preference for attack at C-1 [37–41]. The difference in kinetic mechanism shown by StEH1 in the catalyzed hydrolysis of the 2-MeSO enantiomers indicates that the difference in energy bar- riers for formation of the distinct alkylenzymes is con- siderably smaller when the (R,R)-enantiomer is the substrate. With (S,S)-2-MeSO, the presence of one single (R,S)-diol product suggests that, with this enan- tiomer, only the ES (but not the E¢S) complex in Fig. 3D may be transformed into alkylenzyme and, subsequently hydrolyzed to product. Hence, the ener- getic barrier for formation of the (S,R)-alkylenzyme intermediate resulting from attack at C-2 is consider- ably higher than that required to form the C-1-linked alkylenzyme. In the reaction with (R,R)-2-MeSO, the formation of two diol enantiomers demonstrates that two different alkylenzymes resulting from nucleophilic attack at either C-1 or C-2 of the substrate are stabi- lized, assuming that conformational changes between ES and E¢S do occur. To conclude, the enantiospecificity determined from steady-state kinetic measurements are caused by differ- ent substrate-dependent mechanisms. Although we have only been able to study the individual reaction steps up to the formation of the alkylenzyme interme- diate, it can be deduced from the values of k cat and k 2 that with (S)-SO and TSO, hydrolysis is rate-limiting. With (R)-SO, k cat and k 2 could not be determined, due to poor enzyme saturation within the solubility range of the substrate. Therefore, from the lower-limit values of these parameters (Tables 1 and 2), it cannot be ruled out that the enzyme alkylation rate does contrib- ute to k cat . We propose that with SO and TSO, differ- ences in stabilization of the alkylenzyme are mainly responsible for enantiospecificity. Decreases in K m resulting from rapid formation and ⁄ or slow decay of the alkylenzyme appear to primarily determine selectiv- ity in these cases, whereas tight substrate binding, as in the TSO case, works against enantiodiscrimination. With 2-MeSO, the substrate that is most efficiently discriminated by StEH1, the more complex kinetic mechanism requires other interpretations to under- stand the basis for the enantioselectivity. With this substrate, slow conformational changes limit the over- all catalytic efficiencies. The slow, rate-limiting steps for the (S,S)-enantiomer would escape detection if one was to study the reaction during the steady state, as only one diol enantiomer is produced. The amounts of product formed via either the faster pathway or the slower pathway are additive, and would be detected as an apparent Michaelis–Menten reaction with kinetic parameters described by the dominating faster route. It follows that, for the overall reaction, hydrolysis of the (R,S)-alkylenzyme is rate-limiting with (S,S)-2-MeSO. With (R,R)-2-MeSO, slow conformational changes of the substrate-free enzyme appear to be directly rate- limiting, and therefore serve to mask subsequent cata- lytic steps. Direct analysis of the rates for product enantiomer formation is required for a complete understanding of the reaction scheme in this StEH1- catalyzed transformation. Experimental procedures Materials (R)-SO and (S)-SO (97% and 98% enantiomerically pure, respectively) and (S,S)-MeSO and (R,R)-2-MeSO were pur- chased from Aldrich. (S,S)-TSO and (R,R)-TSO were gifts from P. I. Arvidsson (Department of Biochemistry and Organic Chemistry, Uppsala University). The purities of 2-MeSO and TSO enantiomers were > 99% as judged by chiral HPLC analyses. Protein purification StEH1 was produced in Escherichia coli XL1-Blue cells and purified according to a previously described protocol [10]. The protein concentrations of collected fractions were deter- mined from the absorbance at 280 nm using a molar absor- bance coefficient of 59 030 m )1 Æcm )1 , calculated from the Fig. 4. 13 C-NMR signals of C–O in 1-phenylpropane-1,2-diol (125.7 MHz, CDCl 3 solution) obtained by enzymatic hydrolysis of 2-MeSO enantiomers. (A) Hydrolysis products of (S,S)-2-MeSO with H 2 18 O ⁄ H 2 16 O (1 : 1). (B) Hydrolysis products of (R,R)-2-MeSO with H 2 18 O ⁄ H 2 16 O (1 : 1). See Table 4 for details of relative amounts of isotopologs. Enzyme-catalyzed styrene oxide hydrolysis D. Lindberg et al. 6316 FEBS Journal 275 (2008) 6309–6320 ª 2008 The Authors Journal compilation ª 2008 FEBS amino acid composition. Once purified, the proteins retained full activity upon storage at 4 °C over the time period of analysis. Data for determining steady-state kinetic parame- ters for catalysis of TSO hydrolysis were adapted from [10]. Steady-state kinetics (data collection) The steady-state kinetic parameters of both enantiomers of SO and 2-MeSO were determined by RP-HPLC. Substrates and the internal standard benzyl alcohol were dissolved in acetonitrile and added to the reaction vessel containing 0.1 m sodium phosphate (pH 8.0), to a final concentration of acetonitrile of 1.5% (v⁄ v). The assay pH was chosen to increase the amount of build-up of alkylenzyme intermedi- ate, thereby allowing for determinations of the microscopic rate constants. Alkylenzyme accumulation, at least in the catalyzed hydrolysis of (S,S)-TSO, is linked to a pH-depen- dent decrease in hydrolysis rate and a parallel increase in alkylation rate [13]. Catalyzed reactions were started by the addition of enzyme solution to the reaction vessels. All reactions were carried out at 30 °C, and they were termi- nated at different time points by removing an aliquot of the reaction mixture and injecting it into methanol to reach a final concentration of 37% (v ⁄ v) methanol. Substrate concentrations used were 0.1–0.8, 0.1–0.8, 0.05–0.75 and 0.1–1.2 mm for (S)-SO, (R)-SO, (S,S)-2-MeSO and (R,R)-2- MeSO, respectively, and the enzyme concentrations used were 20, 100, 50 and 200 nm for (S)-SO, (R)-SO, (S,S)- 2-MeSO and (R,R)-2-MeSO, respectively. Using a Water 717 autosampler (SO) or manual injection (2-MeSO) via a Rheodyne 7125 injector, the methanol ⁄ reaction solu- tion mixture was injected into the HPLC system. Reaction constituents were separated on a reverse-phase HighCrom Kromasil C-18 column (25 cm · 3.2 mm diameter), using a Waters 515 pump (flow rate 0.6 mLÆmin )1 ), and the peaks were detected spectrophotometrically with a Waters 484 detector (k = 220 nm). The liquid phase consisted of a 37 : 63 (SO) and 34 : 66 (2-MeSO) mixture of methanol and sodium phosphate buffer (0.1 m, pH 3.0). For the SO enantiomers, the retention times for the reaction constitu- ents and the internal standard were 4.5, 7.1 and 18.4 min (1-phenylethane-1,2-diol, benzyl alcohol, and SO). For the 2-MeSO enantiomers, retention times of 6.9, 8.9 and 45.1 min (1-phenylpropane-1,2-diol, benzyl alcohol, and 2-MeSO) were observed. Pre-steady-state kinetics (data collection) Formation of the alkylenzyme intermediate can be followed by the decrease in the intrinsic Trp fluorescence of the enzyme [10]. Transient-state kinetics were determined in multiple-turnover experiments under pseudo-first-order conditions. Experiments were performed on an Applied Photophysics SX.20MV sequential stopped-flow spectro- photometer at 30 °C, using an excitation wavelength of 290 nm while collecting the fluorescent light through a 320 nm cut-off filter. With all substrates except for (S,S)-2- MeSO, the apparent rate values, k obs , were determined by fitting a single exponential function with floating endpoint, f = A exp()k obs t)+C, to the progression curve; averages of five to eight traces were used in all cases. With (S,S)-2- MeSO, a double exponential function with floating end- point, f = A 1 exp()k obs1 t)+A 2 exp()k obs2 t)+C, was used. The validity of the higher-order equation was verified by F-tests. In both cases, f denotes the averaged progres- sion curve and k obs the averaged observed rates. The ampli- tudes of the fluorescent changes were obtained by subtracting the initial fluorescence value (A) and the float- ing endpoint (C). Substrate concentrations were 6.3–800, 100–1100, 25–1000, 85–1000, 0.8–30 and 12–130 lm [(S)- SO, (R)-SO, (S,S)-2-MeSO, (R,R)-2-MeSO, (S,S)-TSO, and (R,R)-TSO, respectively], using enzyme concentrations of 1, 10, 2, 10, 0.1 and 1.35 lm [(S)-SO, (R)-SO, (S,S)-2-MeSO, (R,R)-2-MeSO, (S,S)-TSO, and (R,R)-TSO, respectively]. Substrate stock solutions were prepared in acetonitrile, after which they, and the enzyme stock solutions, were further diluted in a sodium phosphate buffer (0.1 m, pH 8.0), resulting in a 1–2% (v ⁄ v) final concentration of acetonitrile. Kinetic data analysis The steady-state parameters k cat , K m and k cat ⁄ K m were determined after curve-fitting the Michaelis–Menten equa- tion to the experimental data with mmfit or rffit in simfit (http://www.simfit.man.ac.uk/). The steady-state rate law (Eqn 1) was derived by the method of Waley [42], assuming the kinetic mechanism described in Fig. 1, where K S is k )1 ⁄ k 1 , the dissociation constant of the ES complex, and k 1 is the association rate of enzyme and substrate. The rate constants for alkylenzyme formation, k 2 , together with the dissociation constant for ES complexes, K S , were obtained after fitting Eqn (2) to k obs using qnfit in simfit. The sums of rate constants for alkylenzyme decomposition, k )2 + k 3 , were obtained from the ordinates of fitted curves. Individual values were subsequently extracted from the expression for k cat in the numerator of Eqn (1). ES complexes were assumed to be at equilibrium under the conditions of the measurements. For the slower observed rate with (S,S)-2-MeSO and k obs (R,R)-2-MeSO , data were fitted to Eqn (4) [33,34] using qnfit. 18 O-labeling of 2-MeSO hydrolysis products Initially, control experiments were performed to measure the catalyzed hydrolysis of (R,R)-2-MeSO in the presence of 0.1 m ammonium bicarbonate buffer (pH 8.0), replacing the otherwise used 0.1 m sodium phosphate buffer of the same pH, to establish that reaction rates were compa- rable. For the 18 O-labeling experiment, undiluted 2-MeSO D. Lindberg et al. Enzyme-catalyzed styrene oxide hydrolysis FEBS Journal 275 (2008) 6309–6320 ª 2008 The Authors Journal compilation ª 2008 FEBS 6317 enantiomer was added, 1 lL at a time, to a 0.1 m ammo- nium bicarbonate solution composed of a 1 : 1 (v ⁄ v) H 2 16 O ⁄ H 2 18 O mixture (pH 8.0), 3% (v ⁄ v) acetonitrile, and 1 lm (in the reaction with (S,S)-2-MeSO) or 2.5 lm (in the reaction with (R,R)-2-MeSO) StEH1. The final volume of concentrated epoxide added to the reaction vessel was 7 lL, corresponding to a final concentration of 25 mm diol. The reaction vessel was agitated at 175 r.p.m. and 30 °C, and the progress of the reaction was followed with RP- HPLC until completion. Aliquots of the reaction solution were manually injected using a Rheodyne 7725 injector into a liquid phase consisting of a 50 : 50 mixture of metha- nol ⁄ 0.1 m sodium phosphate (pH 3.0). Product mixture components were separated on a Supelco Ascentis C-18 col- umn (5 lm,25cm· 4.6 mm diameter) coupled to a Shima- dzu Prominence LC-20AD pump. Peaks were detected at 220 nm using a Prominence SPD-M20A diode array detec- tor. After reaction completion, the solvent was evaporated prior to NMR analysis. NMR analysis of 18 O-labeled hydrolysis products NMR spectra were recorded at 500 MHz ( 1 H) and 125.7 MHz ( 13 C), respectively, for CDCl 3 solutions on a Varian Inova spectrometer. Chemical shifts were indirectly referenced to TMS via the solvent signal ( 1 H, 7.26, 13 C, 77.0). Signal assignments were made using gradient- enhanced COSY [43], HSQC [44], and HMBC [45] experi- ments. For diols labeled with 18 O, 13 C-NMR spectra were recorded over a spectral range of 30 kHz, using an acquisi- tion time of 1.3 s, a relaxation delay of 3 s, a pulse flip angle of 26°, and 9188 transients (total experiment dura- tion = 11 h), for a final signal ⁄ noise ratio of 50–60. Data were processed with zero filling to 262 144 points. Relative peak intensities were determined by integration via decon- volution analysis (Lorentzian lineshape) of the spectra, using the manufacturer-supplied software (vnmr 6.1C). Acknowledgements The authors thank M. Engman for establishing the enantiomeric purity of 2-MeSO, A. Gurell for con- structive criticism during manuscript preparation, and J. Gurell for the development of a matlab application. 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Substrate-dependent hysteretic behavior in StEH1-catalyzed hydrolysis of styrene oxide derivatives Diana Lindberg, Adolf Gogoll and Mikael Widersten Department of Biochemistry. precursors in cutin synthesis [4]. The inde- pendence from cofactors in combination with, in some cases, high catalytic efficiencies and enantio- selectivities has created an interest in using epoxide hydrolases