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Abstract Catechol 2,3-dioxygenase [EC 1.13.11.2] from Pseudomonas putida mt-2 (Mpc) catalyzes the extradiol cleavage of catechol to produce 2-hydroxymuconate semialdehyde. The Km values for the catecholic substrate (KmA) and O2 (KmO2), and catalytic constants (kcat) were kinetically determined for eight C3/C4-substituted catechols at 25°C and pH 6.5 or 7.5. The first pKa values (pK1) were determined for eleven catechols (pK1 = 7.26–9.47), correlated with Hammett substituent constants, and electron-withdrawing substituents significantly stabilized the monoanionic species of free catechols. Mpc preferred catechols with non-ionic substituents at the C3 or C4 position. 3-Phenylcatechol, a biphenyl, was cleaved, while 4-tert-butylcatechol was not. The logarithm of kcat/KmA (substrate specificity constant) exhibited a good linear correlation with pK1, with the exception of those for 4-halocatechols. The logarithm of kcat/KmO2 showed a good linear correlation with pK1, with the exception of that of 3-phenylcatechol. These results demonstrate that catechol binding to the Mpc active site, the following O2 binding, and the activation of the bound O2 are all sensitive to electronic effects of the substituents. However, kcat did not correlate significantly with pK1. The present study distinguishes clearly between the electronic and the steric effects of catecholic substrates in the reactivity of Mpc, and provides important insight into the mechanistic basis for a vast range of substrate specificities of extradiol dioxygenases. catechols, dioxygenase, non-heme iron, oxygen, substituent effects, pKa values of catechols. Mpc, catechol 2,3-dioxygenase from Pseudomonas putida mt-2; HOMO, the highest occupied molecular orbital; BphC, 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas sp. KKS102. Received March 31, 2004; accepted April 21, 2004 Many aerobic microorganisms can degrade aromatic compounds to aliphatic compounds using a meta-cleavage pathway (1–3). In the biodegradation, aromatics are first converted to 1,2-dihydroxybenzene (catechol) derivatives, and then the catechols are subjected to oxidative cleavage of the aromatic ring adjacent to the vicinal hydroxyl groups (meta or extradiol cleavage) to yield 2-hydroxy-6-keto-2,4-dienoic acid products (substituted 2-hydroxymuconate semialdehydes) (Fig. 1A). The extradiol catechol dioxygenases catalyze the cleavage using mostly Fe(II) ions or sometimes Mn(II) ions (4–6) as the sole cofactors. A large number of extradiol dioxygenases have been isolated for bioremediation from various aerobic microorganisms, and are classified into two large groups (Types I and II) based on primary and tertiary structural data (7–9). Since the extradiol cleavage step is often a bottleneck step in the complete degradation of aromatic pollutants such as alkylbenzoates and polychlorobiphenyls (PCBs) (10–12), it is important to understand the structural and mechanistic basis of the substrate specificity of this large enzyme family. In natural environments, the availability of O2 is usually limited due to the low solubility and slow diffusion rate of O2 in aqueous solutions. Therefore, the dependence of the enzyme activity on O2 concentration is an especially important factor in the efficiency of the enzymes. The catalytic mechanism of extradiol dioxygenases has been actively investigated. Figure 1 is a summary of proposed reaction mechanisms as reported in recent reviews (13–16). In the reaction, catechol first binds to the active site with bidentate coordination to the Fe(II) ion in a monoanionic manner (17, 18) (Fig. 1B). Then, dioxygen binds directly to the metal center of the enzyme-substrate complex (EA) to form a ternary enzyme-substrate-O2 complex (18) (EAO2) in which O2 is activated by accepting one electron from the bound catechol via the Fe(II) center (19) (Fig. 1C). A recombination in the Fe(II)-superoxide-semiquinone complex results in a proximal hydroperoxide intermediate (20), and then alkenyl migration in the intermediate gives a α-keto lactone and a hydroxide ion bound to the Fe(II) center (21) (Fig. 1D). The requirement of a proton donor for this series of reactions has been proposed based on both experimental and theoretical studies (22, 23). The lactone is attacked by the hydroxide ion to give the opened ring product (EP). Most of the reaction steps proposed for extradiol dioxygenases (Fig. 1) have not been experimentally substantiated, because reaction intermediates other than the EA complex are invisible and not isolatable under physiologically realistic conditions. In addition to this difficulty in the transient kinetics, steady-state kinetic experiments are also difficult due to the extreme instability of the enzymes in the presence of O2. Therefore, in most cases only the apparent values of the catalytic constant (kcat) have been examined, and analyses of steady-state kinetic parameters, including Km for O2 (KmO2), have been carried out for only very few enzymes (12, 24, 25). We describe here the quantitative analysis of the substrate specificity of recombinant catechol 2,3-dioxygenase [EC 1.3.11.2] from Pseudomonas putida mt-2 (metapyrocatechase, Mpc). Using improved assay methods, we were able to measure the kinetic parameters of Mpc for various C3/C4-substituted catechols with exceptional precision compared to other enzymes belonging to the same enzyme family. To discriminate between the electronic and steric effects of the substituents on the Mpc reaction, we determined the first pKa (pK1) of eleven catechols as a quantitative measure of the electronic nature of the substituents. We show that the substrate specificity of Mpc in terms of kcat/KmA and kcat/KmO2 is significantly affected by the electronic nature of the C3- or C4-substituents. Unexpectedly, the kcat values are less sensitive to the electronic nature of the substituents. EXPERIMENTAL PROCEDURES Materials Catechol, 4-bromocatechol, 4-chlorocatechol, 3-chlorocatechol, 3,4-dihydroxybenzyl alcohol (4-hydroxymethycatechol), 3-methycatechol, 4-ethycatechol, 4-nitrocatechol, and 4-tert-butylcatechol were purchased from Tokyo Kasei Kogyo (Tokyo); protocatechualdehyde (4-formylcatechol), 4-methylcatechol and L-ascorbic acid were from Nacalai Tesque (Kyoto); 2,3-dihydroxybiphenyl (3-phenylcatechol), protocatechuic acid (4-carboxycatechol), 3,4-dihydroxyphenylacetate (4-carboxymethylcatechol), and ascorbate oxidase [EC 1.10.3.3] were from Wako Pure Chemical Industries (Osaka); and 2,3-dihydroxybenzaldehyde (3-formylcatechol) was from Sigma-Aldrich Japan (Tokyo). All other chemicals were of analytical grade and used without further purification. Recombinant Mpc was expressed in Escherichia coli, purified to homogeneity, and stored in crystalline form in the presence of 10% (v/v) acetone as described previously (24). Spectroscopic Titration of Catechols Catechols were titrated with HCl and NaOH at 25°C in 0.15 M NaCl under anaerobic conditions according to the method described previously (26). A freshly prepared catechol solution (90–200 µM) was vigorously bubbled with argon for 5 min. An aliquot of the argon-saturated solution (3.0 ml) was transferred to a cuvette. The cuvette was then sealed with a silicone plug and set in a thermo-jacketed cell holder equipped with a magnetic stirrer in a Shimadzu UV-2200 spectrophotometer (Shimadzu, Kyoto). Throughout the titration experiment, moisturized argon was continuously flushed over the surface of the solution via a 19-guage needle through the plug. The argon was allowed to pass out through a thin hole in the plug, through which aliquots of argon-saturated aqueous NaOH or HCl solution (0.5–2.0 µl) were added to the catechol solution with a gas-tight syringe. Spectra were recorded over the wavelength range from 220 nm to 600 nm. The pH was measured using a thin pH electrode (6029–10T, Horiba, Kyoto) inserted through the hole in the plug. The pH-dependence of the observed molar absorption coefficient (ε) at the absorption peak of the monoanionic species of a catechol was fitted to Eq. 1 by nonlinear regression, ε = (εH + ε–10pH–pK1)/(1 + 10pH–pK1) (1) where pK1 is the first pKa of a catechol, εH and ε– are the molar absorption coefficients of its neutral and monoanionic species, respectively. Handling of Mpc Samples Mpc is a homotetramer and each subunit has a single active site in the C-terminal-domain (24, 27). No substantial cooperativity has been found among the active sites. Therefore, hereafter, the enzyme concentration is always described as the Mpc subunit concentration. The enzyme subunit concentrations were determined spectrophotometrically using a molar absorption coefficient of 43,830 M–1 cm–1 at 280 nm (24). A concentrated Mpc solution was prepared as follows. Mpc crystals were collected from an aliquot of a crystalline Mpc stock solution stored at 4°C (about 200 µl, specific activity ≥ 300 U/mg) by centrifugation, and dissolved in a minimum amount of 50 mM HEPES, pH 7.5, ionic strength = 0.15 M adjusted with NaCl (HEPES buffer). The concentration of this concentrated Mpc stock solution was 1–2 mM and its specific activity remained greater than 100 U/mg for several weeks at 4°C under aerobic conditions. A dilute Mpc stock solution (2–6 µM) for steady-state kinetic experiments was prepared in a sealed vial in the presence of an O2-eliminating system composed of l-ascorbate and ascorbate oxidase. About 2 mg of solid l-ascorbic acid and 3 ml of argon-saturated HEPES buffer were placed into a 3.0-ml glass vial (Maruemu, Osaka). After sealing the vial with a PTFE/butyl rubber septum and aluminum cap using a clamp, the ascorbic acid was completely dissolved by gentle rotation of the vial. Then, an aliquot of ascorbate oxidase solution (10 µl, 20 U) was added to the vial with a micro-syringe through the septum. After incubation for more than 5 min at room temperature, an aliquot of the concentrated Mpc solution (5–10 µl) was added to the vial through the septum with a micro-syringe. The activity of the resultant dilute Mpc stock solution (50–170 U/mg) was stable for several days, and no significant difference in the enzyme activity was found before and after a series of kinetic experiments. Kinetic Assays Kinetic experiments were carried out at 25°C in 50 mM MES, pH 6.5, ionic strength = 0.15 M and in 50 mM HEPES, pH 7.5, ionic strength = 0.15 M. The dependence of the initial velocity on the O2 concentration was measured in 2.9 ml of reaction mixture containing a constant concentration of a catecholic substrate (10–100 fold higher than the apparent Km value determined for the substrate under air-saturation) by following the consumption of O2 using a Model 5331 Clark-type polarographic O2 electrode (Yellow Springs Instrument, Yellow Springs, OH, USA). The O2 concentration was controlled over the range of 1–1,000 µM as described previously (26). The signal from the O2 electrode was recorded with a Unicorder U-228 analogue recorder equipped with an accurate amplifier (Pantos, Kyoto). The signal was calibrated by recording the rapid and complete cleavage of a known amount of a relevant substrate in a specified buffer with a small aliquot of the concentrated Mpc stock solution (0.5–2.0 µl). The 1:1 stoichiometry of O2 consumption and product formation for each substituted catechol was confirmed by comparing the calibration signal with the signal recorded for the complete cleavage of a known amount of catechol. The dependence of the initial velocity on catecholic substrate concentration was measured in 3.0 ml of reaction mixture under air-saturation ([O2] = 240–260 µM) by following the formation of the product using a UV-140 UV/VIS spectrophotometer (Shimadzu, Kyoto) equipped with a thermo-jacketed cell holder. The signal from the spectrophotometer was recorded with a Unicorder U-228 analogue recorder, and the signal was calibrated as described above for the calibration of the signal of the O2 electrode. The enzymatic reaction was started by the addition of a small aliquot of the dilute Mpc stock solution (0.5–5 µl), which was removed from the vial with a micro-syringe just before addition. To determine the value of the catalytic constant (kcat) of Mpc, the amount of the active ferrous center (Et) should be known for the relevant kinetic assay conditions. Et was estimated as follows. First, using the same amount of dilute Mpc stock solution as used for the kinetics experiments, the activity was measured at 25°C in 50 mM HEPES buffer (pH 7.5, ionic strength = 0.15 M) containing 100–200 µM catechol under air-saturation (standard assay conditions). Because there is a linear relationship between iron content and specific activity (24), the Et value was calculated by multiplying the total Mpc subunit concentration by the ratio of the measured specific activity and the specific activity of fully active Mpc. We assumed that fully active Mpc shows a specific activity of 477 U/mg (24). Analysis of Steady-State Kinetic Data Steady-state kinetic data were analyzed using Eq. 2 on the basis of an ordered Bi Uni mechanism in which the binding of the catecholic substrate, A, precedes that of O2 (Fig. 1A), v = kcatEtAO2/(KdAKmO2 + KmAO2 +KmO2A + AO2) (2) where v is the initial velocity, KmA and KmO2 represent the Km for catecholic substrate and O2, respectively, KdA represents the dissociation constant for the binding of the substrate to the enzyme, A and O2 in italic letters are the molar concentrations of the substrate and O2, respectively. Et is the molar concentration of the active ferrous center. Based on Eq. 2, the dependence of the initial velocity on the catechol concentration under fixed O2 concentration and on the O2 concentration under a fixed catechol concentration is described by the following Michaelis-Menten equations, respectively. v = VmaxappA/(KmAapp + A) (3) v = VmaxappO2/(KmO2app + O2) (4) The relations between the actual and apparent values of Vmax and Km are summarized in Appendix. All kinetic data obtained fitted well to Eq. 3 or 4. We first determined the apparent Vmax and Km values by linear regression to s/v versus s plots with equal weighting, where s is the concentration of the catecholic substrate or O2. Then, to confirm the goodness of the fit, the data were plotted on the theoretical v versus s curve drawn using the fitted values of Vmax and Km. pH-Dependence of kcat Values The effects of pH on the kcat of Mpc for catechol and 4-chlorocatechol were examined using the following buffers: 50 mM MES-NaOH (pH 5.5–6.5), 50 mM HEPES-NaOH (pH 6.5–8.0), 50 mM Tris-HCl (pH 8.0–8.5), 50 mM glycine-NaOH (pH 9.0–10.0), 50 mM CHES-NaOH (pH 9.0–10.0), and 50 mM CAPS-NaOH (pH 10–11). The ionic strength of all buffers was adjusted to 0.15 M with NaCl. The relative kcat values of Mpc for catechol, 4-methylcatechol, 4-chlorocatechol, 4-bromocatechol, 4-formylcatechol, and 3-formylcatechol were measured using saturating concentrations of catecholic substrates in air-saturated MES (pH 6.5), HEPES (pH 7.5), and Tris-HCl (pH 8.5) buffers and the same amount of Mpc (the final Mpc concentration of 11.2 nM, Et = 4.73 nM). The pH-dependence of the kcat values for catechol and 4-chlorocatechol was simulated with Eq. 5 supposing that the Mpc-substrate-O2 ternary complex has three ionizable groups involved in the rate-determining step or steps, kcat = (kcat,110pKII–pH + kcat,2)/(1 + 10pKII–pH + 10pKI + pKII–2pH + 10pH–pKIII) (5) where KI,KII, and KIII (KI > KII > KIII) are the respective dissociation constants of the three ionizable groups of the ternary complex, and kcat,1 and kcat,2 are the catalytic constants for the singly and doubly deprotonated forms of the ternary complex, respectively. RESULTS Properties of Substituted Catechols Upon titration over the pH range 3.5–11, the absorbance due to the lowest energy transition of each catechol showed a red shift (11.5–83.6 nm) and an increase in absorption as the pH increased. Isosbestic points were observed for the absorption band of all catechols examined except 3-methylcatechol and 4-nitrocatechol. In this pH range, we did not observed any tendency for further deprotonation of the monoanionic species to the dianionic species for any catechol. The observed reversible pH-dependent changes in the absorption fitted Eq . 1 well. The pK1 values obtained are summarized in Table 1 together with the λmax values of the lowest energy transition of the neutral and monoanionic species of catechols. Electron-withdrawing substituents decreased the pK1 of catechol (9.32) maximally to 7.26. The pK1 values for C4-substituted catechols showed the best correlation with the mean of the σp– and σm values (28) (ρ = –2.55, Fig. 2A), while those of the C3-substituted catechols showed a good correlation with σm values (ρ = –3.77, Fig. 2B). These results suggest that the monoanion of a C4-substituted catechol exists in equilibrium between the 1-hydroxy-2-phenolate- and 2-hydroxy-1-phenolate-forms of the catechol, and that the monoanion of a C3-substituted catechol exists predominantly in the 2-hydroxy-1-phenolate form. The obtained ρ value for C4-substituted catechols is comparable to that reported for the linear relationship between the pKa values of C4-substituted phenols and Hammett σp– values (ρ = –2.23 in H2O and = –2.67 in 50% ethanol) (29). Steady-State Kinetic Analyses 3-Chlorocatechol, 4-ethylcatechol, and 4-hydroxymethycatechol rapidly inactivated Mpc during turnover, although significant amounts of products were formed before complete inactivation of Mpc. Therefore, we could not obtain reliable kinetic parameters for these three catechols. 4-Nitrocatechol was cleaved by Mpc, but the KmO2 value was very large (more than 1 mM), and we could not obtain precise kinetic parameters for this catechol. Catechols with anionic substituents such as 3,4-dihydroxybenzoate (4-carboxycatechol) and 3,4-dihydroxyphenylacetate (4-carboxymethylcatechol) were not cleaved by Mpc. The enzyme could not cleave 4-tert-butylcatechol, while 2,3-dihydroxybiphenyl (3-phenylcatechol) was a relatively good substrate of Mpc. Therefore, we carried out detailed kinetic analyses for the eight catechols listed in Table 2 at pH 6.5 and 7.5. Figure 3 shows typical kinetic data obtained in the present study. All kinetics data fitted the Michaelis-Menten equation well. The best-fit steady-state kinetics parameters are provided in Table 2. The KmA value of Mpc was mostly in the range of 0.2–3 µM; for 4-formylcatechol only it was 15.1 and 22.8 µM at pH 6.5 and 7.5, respectively. On the other hand, the KmO2 values of Mpc were in the range of 3–20 µM except for catechols with formyl or phenyl substituents. For 4-formylcatechol the KmO2 value was 492 and 313 µM at pH 6.5 and 7.5, respectively. For 3-formylcatechol the KmO2 value was 259 µM at pH 6.5, whereas it was 25 µM at pH 7.5. For 3-phenylcatechol the KmO2 value was 190 µM at pH 6.5. Because the O2 concentration is 240–260 µM in air-saturated buffers at 25°C, the apparent KmA values obtained under air-saturation for these catechols with high KmO2 values were possibly not good approximations of the actual values (Eq. A3 in “appendix”). The largest kcat value was found to be 402 s–1 for catechol at pH 6.5, and the smallest was 24 s–1 for 3-phenylcatechol at pH 7.5. The difference in the kcat values of Mpc among eight catechols examined was maximally about 17-fold, while the largest differences in the KmA and KmO2 values were 88- and 129-fold, respectively. Specificity Constants as a Function of Catecholic pK1 The specificity constant kcat/KmA of Mpc was maximally about 2.0 × 108 M–1 s–1 for 4-methylcatechol and 4-chlorocatechol, slightly greater than that for catechol (about 1.5 × 108 M–1 s–1, Table 2). The smallest kcat/KmA was 4.9 × 106 M–1 s–1 for 4-formylcatechol. Figure 4A shows the dependence of the logarithm of the relative kcat/KmA on pK1. With exception of 4-chlorocatechol, there was a good linear correlation between log kcat/KmA and pK1 (slope = 0.66, r2 = 0.96) at pH 7.5. The data point obtained for 3-phenylcatechol, a bulky bicyclic catechol, was on the line. A good linear correlation was also found between log kcat/KmA and pK1 (slope = 0.48, r2 = 0.94) at pH 6.5 when the data for 4-chloro- and 4-bromo-catechols were excluded. According to the reaction mechanism illustrated in Fig. 1A, the specificity constant of kcat/KmA is equal to the binding rate constant of the catecholic substrate to the enzyme active site (k1, see Eq. A7 in Appendix). Therefore, the results indicate that electron-withdrawing substituents reduce the binding rate of catechol. The catechol binding process seems to be less sensitive to steric factors, such as the position of the substitution (C3 or C4) and the bulkiness of the substutients. Chlorine and bromine substituents at the C4 position showed halogen-specific effects to compensate for their electron-withdrawing effects on the substrate binding process. It is well known that 3-halocatechols rapidly inactivate many extradiol dioxygenases including Mpc in a mechanism-based manner (30–32). There may be some common mechanism in which chlorine and bromine interact with the active site of Mpc to induce halogen-specific effects. The kcat/KmO2 values decreased in the order 4-methycatechol > catechol > 4-chlorocatechol > 4-bromocatechol > 4-formylcatechol > 3-formylcatechol at pH 6.5. The plot of the logarithm of the relative kcat/KmO2versus pK1 revealed a good linear correlation (slope = 0.57, r2 = 0.91) at pH 6.5 without any outlier (Fig. 4B). At pH 7.5, the kcat/KmO2 values decreased in the order catechol > 4-methylcatechol > 3-methylcatechol > 4-chlorocatechol > 3-formylcatechol > 4-formylcatechol > 3-phenylcatechol. With exception of 3-phenylcatechol, there was a good linear correlation between log kcat/KmO2 and pK1 (slope = 0.83, r2 = 0.93) at pH 7.5. The aberrant behavior observed for 3-phenylcatechol suggests that bulky substituents at the C3 position inhibit sterically the binding of O2. For all other catechols, the specificity constant kcat/KmO2 decreased as the electron-withdrawing nature of the substituents increased. The specificity constant of kcat/KmO2 is proportional to the rate constant of O2 binding to the EA complex (k2) and intimately related to the activation of the bound O2 (Fig. 1A and Eq. A8 in Appendix). The ternary enzyme-catechol-O2 complex is committed to ring cleavage (k3) or dissociates O2 (k–2). When k–2 is much smaller than k3 or of the same order of magnitude as k3, then kcat/KmO2 reflects mainly the rate constant of O2 binding (k2). The observed linear free energy relationship suggests that electron-withdrawing substituents suppress the binding of O2 molecules to the enzyme-catechol complex. Substituent Effects on the Overall Reaction Rate Figure 5 shows the plot of the logarithm of the relative kcat versus pK1. Although there seems to be a weak tendency for the kcat value to decrease as the pK1 value decreases, no clear correlation between the kcat and pK1 values was observed. The results suggest that after the proper activation of O2 in the enzyme active site, the irreversible steps to the enzyme-product complex (Fig. 1D) are less sensitive to the electronic nature of the substituent compared to the preceding two steps of catechol and O2 binding. pH-Dependence of the kcat Values for Catechol and 4-Chlorocatechol Substituents effects on the kcat values were further examined as a function of pH. The kcat of Mpc for six catechols was determined at pH 6.5, 7.5, and 8.5 (Table 3). The kcat value for 4-methylcatechol did not changed as pH increased from 6.5 to 8.5 and remained about 250 s–1, while the kcat values for catechol, 4-formylcatechol, and 3-formylacatechol decreased as the pH increased from 6.5 to 8.5. On the other hand, the kcat value for 4-chlorocatechol and 4-bromocatechol increased as the pH increased from 6.5 to 8.5. To investigate the different pH dependences of the kcat values, those for catechol and 4-chlorocatechol were measured over the pH range of 5–10. The results are shown in Fig. 6. The kcat values at pH < 5.5 could not be measured due to rapid enzyme inactivation, and those at pH > 10.5 could not be measured because significant autooxidation of catechols occurs at high pH values. The pH dependence of the kcat values of the two catechols could be simulated with a triple ionization curve (Eq. 5) using the same set of three pKa values of 5.5, 7.5, and 9.8. Since the pH profile of kcat reflects the ionization state of the ternary EAO2 complex, the results suggest that a deprotonated group with an approximate pKa value of 5.5 and a protonated group with an approximate pKa value of 9.8 in the EAO2 complex are essential for ring cleavage. In addition to these two groups, it is suggested that another ionizable group with a pKa of 7.5 in the ternary complex affects the rate-determining step relatively weakly in a substrate-dependent manner; deprotonation of this group disfavors the enzymatic cleavage of catechol but facilitates the cleavage of 4-chlorocatechol. DISCUSSION In the present study, we show for the first time that the electronic nature of substituents is a major factor in determining the specificity constants of kcat/KmA and kcat/KmO2 of Mpc for a series of C4/C3-substituted catechols whose pK1 values range from 7.26 to 9.47. Exceptions are 4-halocatechols in the case of kcat/KmA, and 3-phenylcatechol in the case of kcat/KmO2. The overall turnover rate (kcat) of Mpc is less sensitive to the electronic nature of substituents than the specificity constants. Cleaving the Catechol Ring There have been two opposing proposals for the electronic effects on the irreversible ring cleavage step of the enzymatic reaction (EAO2→EP). One considers that electron-withdrawing groups on the catechol ring support the nucleophilic attack of the O2 molecule on the C3 carbon atom to form a peroxy intermediate (13, 33). The other considers that a peroxy intermediate is formed by a radical mechanism (see Fig. 1D) and that electron-withdrawing substituents inhibit the alkenyl migration in the peroxy intermediate (22). If these reaction processes are the rate-determining step in the overall reaction, then electron-withdrawing substituents are expected to increase or decrease the kcat value, respectively. However, we could not observe a significant correlation between kcat and pK1. The pH dependence of the kcat of Mpc suggests the existence of an ionizable residue with a pKa of about 7.5 that is not essential for ring cleavage but affects the overall reaction rate. Thus, the present results on kcat values suggest that steric and/or group-specific factors of the substituents significantly affect the irreversible ring cleavage step to obscure the electronic effects. Of course, there is a possibility that the release of the product from the enzyme-product complex is the rate-determining step in the overall reaction and determines the kcat value. Experimental determination of the rate of product release (k4) is needed, but it is difficult to measure the k4 value because it is difficult to discriminate spectrophotometrically between the EP complexes and free products. Substrate Binding Surprisingly, there is a good linear free energy relationship between the kcat/KmA of Mpc and the pK1 of catechols, including 3-phenylcatechol. Because the specificity constant of kcat/KmA is equal to the rate constant of the binding of substrate to the enzyme (k1), this result suggests that the Mpc active site is large enough to accommodate these substituents at the C4 or C3 position, and that a substituent at the C4 position that is smaller than or similar to a formyl group does not give rise to steric hindrance with chemical groups in the active site, but a substituent at the C4 position that is larger than or similar to a nitro group gives rise to significant steric hindrance for the binding of catechol derivatives to the Mpc active site. It is intriguing that electron-withdrawing substituents decrease the substrate binding rate of Mpc. Mono-substituted phenols are competitive inhibitors of Mpc for catecholic substrates, and electron-withdrawing substituents increase the stability of the enzyme-phenol complex (our unpublished results). One possible mechanism to explain the electronic effects on the catechol binding rate is as follows. The binding of catechol to the active site consists of at least two steps. First, an intermediate is formed in which the catechol is monodentately bound to the Fe(II) center, as with phenols. The intermediate is stabilized by electron-withdrawing substituents, and the transition from the intermediate to the final EA complex is the rate-determining step in the substrate binding. To test this possible mechanism for substrate binding, the substituent effects on the binding energy between catecholic substrates and the enzyme are now investigated in our laboratory. O2 Binding and Activation A significant linear free energy relationship was found between the specificity constant of kcat/KmO2 for Mpc and pK1. The HOMO energies (Ehomo) of free catecholate monoanions have been calculated for catechol and 4-methyl-, 4-chloro- and 4-bromo-catechols (34). We found a linear relationship between the reported Ehomo (eV) and pK1 values with slope of 0.41. The HOMO energy of the catecholate monoanion bound to the active site Fe(II) center is expected to be reduced by electron-withdrawing substituents. If one electron transfer from the bound catechol monoanion to the O2 via the Fe(II) center is the rate-determining step in the O2 binding, then electron-withdrawing substituents inhibit O2 binding by reducing the nucleophilicity of the catechol in the active site. Because kcat/KmO2 is proportional to the rate of O2 binding to the EA complex (k2), the observed electronic effects on the kcat/KmO2 value is possibly due to effects on the k2 value. Comparison of Substituent Effect between Intradiol- and Extradiol-Cleaving Enzymes Intradiol-cleaving enzymes catalyze the cleavage of catechols between the vicinal hydroxyl groups using Fe(III) ions. The effects of C4- and/or C3-substituents on the kcat values have been investigated for intradiol dioxygenases (34, 35). A linear correlation between the logarithm of the kcat values and Hammett σ values has been found for pyrocatechases I and II (35). A strong linear correlation between the logarithm of the kcat values and the HOMO energies of C4-substituted catechol monoanions has been reported for catechol 1,2-dioxygenase from Pseudomonas putida (arvilla) C1 (34). It is considered that O2 molecules cannot bind directly to the Fe(III) center of the enzyme-catechol complex (13, 15). The attack of O2 on the activated substrate to form the ternary enzyme-substrate-O2 complex (peroxy intermediate) and subsequent acyl migration of the intermediate is proposed as the rate-determining step in intradiol cleavage (15, 22, 34). In the case of Mpc, we did not observe significant electronic effects of substituents on the kcat values. The present results suggest that in the ternary Mpc-catechol-O2 complex the bound O2 molecule is activated to a superoxide species prior to the formation of the alkyl-peroxy intermediate. In the case of intradiol dioxygenases, the electrophilic attack of O2 on the activated substrate includes the activation of O2 to a peroxy species. Thus, the electronic effects on the kcat of intradiol dioxygenases may correspond to those on the kcat/KmO2 of Mpc. Active Site Structure Crystal structures of Mpc in complexes with substituted catechols are essential for interpreting the present results, but are still not available despite our continued efforts. For 2,3-dihydroxybiphenyl 1,2-dioxygenass from Pseudomonas sp. strain KKS102 (BphC), a high-resolution structure of the BphC-2,3-dihydroxybiphenyl-NO complex has recently been determined (18). Since 2,3-dihydroxybiphenyl (=3-phenylcatechol) is a moderately good substrate for Mpc and the subunit structure of Mpc resembles that of BphCKKS102, as described previously (27), the active-site structure of the Mpc-acetone complex was compared to that of the BphC-2,3-dihydroxybiphenyl-NO complex (Fig. 7). The molecular plane of acetone, a competitive inhibitor of catechol (24), is parallel to the imidazole ring of His246 (Fig. 7A), just as the catechol ring of the bound substrate is parallel to the imidazole ring of His240 (Fig. 7B). The space occupied by the 3-phenyl group of the substrate bound to the BphC active site is wide open at the corresponding position in the Mpc active site, consistent with the fact that Mpc can catalyze the extradiol cleavage of 3-phenylcatechol. On the other hand, BphC cannot catalyze the extradiol cleavage of 4-substituted catechols (manuscript in preparation). In fact, the C4 atom of the catechol ring is in contact with the side chain of Thr280 and cannot accept even relatively small substituents such as chlorine, whereas there is a relatively wide space to accept C4-substituents in the corresponding region of the Mpc active site. In fact, the binding of 4-formylcatechol to the Mpc active site is sterically well tolerated. Steric hindrance becomes apparent for the binding of 4-nitrocatechol, and bulky t-butyl groups at the C4 position can not be accepted in the Mpc active site. The pH-dependence of the kcat values (Fig. 6) revealed that three ionizable residues of the Mpc-substrate-O2 complex are catalytically important. There are only three ionizable residues at the active site of Mpc other than the three Fe(II) ligands; His199, His246, and Tyr255 (Fig. 7A). On the basis of the structural data for the BphCKKS102-substrate complex (18), BphC-substrate-NO complex (18), and BphCLB400-substrate complex (17), the following is suggested: The residue with a pKa value of 5.5 is His199, and acts as a proton shuttle between the 1-OH group of the substrate and the proximal oxygen atom of the activated O2 (Fig. 1, B and C). The residue with a pKa value of 7.5 is His246, and affects the rate-determining step through a steric mechanism. (Fig. 1D). Tyr255 is a probable candidate for the residue with a pKa value of 9.8, and forms a hydrogen bond with the 2-OH group of the catehol to contribute to the asymmetric binding of the substrate that is essential for the binding and activation of O2 (Fig. 1, C and D). In the ternary complex of the BphC-substrate-NO complex, the NO binding site (=O2 binding site) is formed by the catechol ring of the substrate, the side chains of Phe186, Val147, Ala197 and His194, and iron-chelating His145 and His209 (18). In the Mpc active site, the Val147 of BphC is replaced by the more bulky Leu155, and the other residues are completely conserved. Therefore, it is expected that the C4-substituent of catecholic substrate does not contact the O2 binding site of Mpc directly. On the other hand, the bulky C3-substituent may hinder O2 binding sterically. In fact, only 3-phenylcatechol shows an aberrant low kcat/KmO2 values, and the electronic effects of the substituents on kcat/KmO2 were successfully observed without disturbance by steric effects. Concluding Remarks The reaction mechanisms so far discussed have been based only on the apparent kcat values. In the present study, we determined kinetically the true values of kcat, KmA, and KmO2 for various catecholic substrates for the first time among extradiol dioxygenases, and analyzed the substrate specificity constants and the overall reaction rate in terms of the pK1 values of catecholic substrates, i.e., the electronic nature of the substrates. All the processes of catechol binding to the Mpc active site, the subsequent O2 binding, and the activation of the bound O2 are sensitive to the electronic effects of the substituents. However, the overall reaction rate (kcat) does not correlate significantly with pK1. The present study distinguishes clearly between the electronic and steric effects of catecholic substrates on the reactivity of Mpc, and sheds new light on the mechanistic basis for the different substrate specificities of extradiol dioxygenases. To clarify further the reaction mechanisms of extradiol-type dioxygenases, the following investigations are now under way: 1) examination of substituent effects on the reaction process and the energy of Mpc-substrate complex formation, 2) kinetic and crystallographic studies of BphC (2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas sp. KKS102) using C3/C4-substituted catechols. APPENDIX The dependence of the initial velocity (v) on the catecholic substrate concentration (A) at a constant concentration of O2 (O2) is expressed as follows: v = kcatappEtA/(KmAapp + A) (A1) kcatapp = kcatO2/(KmO2 + O2) (A2) KappmA = (KdAKmO2 + KmAO2)/(KmO2 + O2) (A3) The dependence of the initial velocity on the O2 concentration at a constant concentration of catechol is expressed as follows: v = kcatappEtO2/(KmO2app + O2) (A4) kcatapp = kcatA/(KmA + A) (A5) KmO2app = KmO2(KdA + A)/(KmA + A) (A6) To obtain actual kcat values, the dependence of the initial velocity on the O2 concentration at saturating concentrations of catecholic substrate should be examined (Eq. A5). When KmO2 is larger than the O2 concentration in air-saturated buffer, the apparent kcat value obtained by examining the dependence of the initial velocity on the catechol concentration using air-saturated buffer is approximately equal to (kcat/KmO2)O2. The following are the relationships between the steady-state kinetic parameters and the rate constants described in Fig. 1: k1 =kcat/KmA (A7) (A8) (A9) This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (B, NO. 753) (NO. 13125202) (to K.H., T. I., and H. T.) and a Grant-in-Aid for Scientific Research (C) (NO. 15590485) (to T. I., and H. T.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. * To whom correspondence should be addressed. Tel: +81-77-548-2158, Fax: +81-77-548-2157, E-mail: teishida@belle.shiga-med.ac.jp Open in new tabDownload slide Fig. 1. A summary of reaction mechanisms proposed for extradiol catechol dioxygenases. (A) The reaction cycle of enzymatic cleavage of catechols. The relationship between the rate constants and steady-state kinetic parameters are given in Appendix. X denotes a substituent. (B) A schematic active site structure of the EA complex (see Fig. 7). (C) An O2 activation mechanism in the EAO2 complex. (D) Proposed intermediates for the conversion from the EAO2 to EP complex. The rate constant k3 in Fig. A has the following relation to the elementary rate constants shown in the figure: 1/k3 = 1/kα + 1/kβ + 1/kγ + 1/kδ. Open in new tabDownload slide Fig. 1. A summary of reaction mechanisms proposed for extradiol catechol dioxygenases. (A) The reaction cycle of enzymatic cleavage of catechols. The relationship between the rate constants and steady-state kinetic parameters are given in Appendix. X denotes a substituent. (B) A schematic active site structure of the EA complex (see Fig. 7). (C) An O2 activation mechanism in the EAO2 complex. (D) Proposed intermediates for the conversion from the EAO2 to EP complex. The rate constant k3 in Fig. A has the following relation to the elementary rate constants shown in the figure: 1/k3 = 1/kα + 1/kβ + 1/kγ + 1/kδ. Open in new tabDownload slide Fig. 2. Linear free energy relationships for substituted catechols. The pK1 values were determined at 25°C in 0.15 M NaCl (Table 1). (A) Correlation between pK1 of C4-substituted catechols and (σp– + σm)/2. (B) Correlation between pK1 of C3-substituted catechols and σm. The σ values are taken from Ref. 28. Open in new tabDownload slide Fig. 2. Linear free energy relationships for substituted catechols. The pK1 values were determined at 25°C in 0.15 M NaCl (Table 1). (A) Correlation between pK1 of C4-substituted catechols and (σp– + σm)/2. (B) Correlation between pK1 of C3-substituted catechols and σm. The σ values are taken from Ref. 28. Open in new tabDownload slide Fig. 3. Typical steady-state kinetic behavior of Mpc reactions with catechols. The experiments were performed using 50 mM MES, pH 6.5, ionic strength = 0.15 M (adjusted with NaCl), at 25°C. (A) The dependence of the initial velocity of the Mpc reaction with 4-methylcatechol (fixed concentration of 179 µM) on O2 concentration. The inset is an enlargement of the data in the low O2 concentration region. The total concentration of active enzyme subunit used was 2.96 nM. The line represents the best fit of the data to the Michaelis-Menten equation (KmO2 = 3.80 ± 1.8 µM, Vmax = 16.4 ± 0.3 µM/min). (B) The dependence of the initial velocity on the concentration of 4-chlorocatechol under air-saturation ([O2] = 240–260 µM). The inset is an enlargement of the data in the low substrate concentration region. The total concentration of active enzyme subunit used was 0.76 nM. The line represents the best fit of the data to the Michaelis-Menten equation (KmA = 0.79 ± 0.52 µM, Vmax = 1.89 ± 0.01 µM/min). Open in new tabDownload slide Fig. 3. Typical steady-state kinetic behavior of Mpc reactions with catechols. The experiments were performed using 50 mM MES, pH 6.5, ionic strength = 0.15 M (adjusted with NaCl), at 25°C. (A) The dependence of the initial velocity of the Mpc reaction with 4-methylcatechol (fixed concentration of 179 µM) on O2 concentration. The inset is an enlargement of the data in the low O2 concentration region. The total concentration of active enzyme subunit used was 2.96 nM. The line represents the best fit of the data to the Michaelis-Menten equation (KmO2 = 3.80 ± 1.8 µM, Vmax = 16.4 ± 0.3 µM/min). (B) The dependence of the initial velocity on the concentration of 4-chlorocatechol under air-saturation ([O2] = 240–260 µM). The inset is an enlargement of the data in the low substrate concentration region. The total concentration of active enzyme subunit used was 0.76 nM. The line represents the best fit of the data to the Michaelis-Menten equation (KmA = 0.79 ± 0.52 µM, Vmax = 1.89 ± 0.01 µM/min). Open in new tabDownload slide Fig. 4. Substituent effects on the specificity constants of Mpc at 25°C. (A) Plot of the logarithm of the relative kcat/KmA at pH 6.5 (solid circles) and 7.5 (open circles) against pK1. The (kcat/KmA)x and (kcat/KmA)H denote the kcat/KmA values for substituted catechol and catechol, respectively. (B) Plot of the logarithm of the relative kcat/KmO2 at pH 6.5 (solid circles) and 7.5 (open circles) against the pK1. The (kcat/KmO2)x and (kcat/KmO2)H denote the kcat/KmO2 values for substituted catechol and catechol, respectively. 1, catechol; 2, 4-methylcatechol; 3,3-methylcatechol; 4,4-chlorocatechol; 5,4-bromocatechol; 6,3-formylcatechol; 7,4-formylcatechol; 8,3-phenylcatechol. Open in new tabDownload slide Fig. 4. Substituent effects on the specificity constants of Mpc at 25°C. (A) Plot of the logarithm of the relative kcat/KmA at pH 6.5 (solid circles) and 7.5 (open circles) against pK1. The (kcat/KmA)x and (kcat/KmA)H denote the kcat/KmA values for substituted catechol and catechol, respectively. (B) Plot of the logarithm of the relative kcat/KmO2 at pH 6.5 (solid circles) and 7.5 (open circles) against the pK1. The (kcat/KmO2)x and (kcat/KmO2)H denote the kcat/KmO2 values for substituted catechol and catechol, respectively. 1, catechol; 2, 4-methylcatechol; 3,3-methylcatechol; 4,4-chlorocatechol; 5,4-bromocatechol; 6,3-formylcatechol; 7,4-formylcatechol; 8,3-phenylcatechol. Open in new tabDownload slide Fig. 5. Substituent effects on the overall reaction rate at 25°C. The logarithm of the relative kcat at pH 6.5 (solid circles) and 7.5 (open circles) was plotted against pK1. The (kcat)H and (kcat),X denote the kcat for catechol and 4-substituted catechols, respectively. The numbers show the same catechol compounds as indicated in Fig. 4. Open in new tabDownload slide Fig. 5. Substituent effects on the overall reaction rate at 25°C. The logarithm of the relative kcat at pH 6.5 (solid circles) and 7.5 (open circles) was plotted against pK1. The (kcat)H and (kcat),X denote the kcat for catechol and 4-substituted catechols, respectively. The numbers show the same catechol compounds as indicated in Fig. 4. Open in new tabDownload slide Fig. 6. pH Dependence of the kcat value of Mpc with catechol and 4-chlorocatechol at 25°C. The thick and thin lines were drawn according to Eq. 5 for catechol (solid circles) and 4-chlorocatechol (open circles) using the values pKI = 5.5, pKII = 7.5, pKIII = 9.8, and the following respective values: kcat,1 = 402 s–1 and kcat,2 = 128 s–1 for catechol; kcat,1 = 150 s–1 and kcat,2 = 248 s–1 for 4-chlorocatechol (see Table 3). Open in new tabDownload slide Fig. 6. pH Dependence of the kcat value of Mpc with catechol and 4-chlorocatechol at 25°C. The thick and thin lines were drawn according to Eq. 5 for catechol (solid circles) and 4-chlorocatechol (open circles) using the values pKI = 5.5, pKII = 7.5, pKIII = 9.8, and the following respective values: kcat,1 = 402 s–1 and kcat,2 = 128 s–1 for catechol; kcat,1 = 150 s–1 and kcat,2 = 248 s–1 for 4-chlorocatechol (see Table 3). Open in new tabDownload slide Fig. 7. Stereo view of the active-site structures of (A) the Mpc-acetone complex (PDB code: 1MPY) (27) and (B) the BphC-DBP-NO complex (PDB code: 1KW8) (18). BphC, 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas sp. strain KKS102; DBP, 2,3-dihydroxybiphenyl. The bound substrates and the side chains are darkly shaded. Small black spheres are Fe(II) ions. These figures were prepared using the program RASMOL (36). Open in new tabDownload slide Fig. 7. Stereo view of the active-site structures of (A) the Mpc-acetone complex (PDB code: 1MPY) (27) and (B) the BphC-DBP-NO complex (PDB code: 1KW8) (18). BphC, 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas sp. strain KKS102; DBP, 2,3-dihydroxybiphenyl. The bound substrates and the side chains are darkly shaded. Small black spheres are Fe(II) ions. These figures were prepared using the program RASMOL (36). Table 1. Properties of C4/C3-substituted catechols. Substituent pK1a Wavelengthb (nm) Neutral form Monoanion H 9.32 ± 0.02 274.8 288.4 4-CH3 9.47 ± 0.01 279.8 294.8 4-CH2OH 8.91 ± 0.03 279.3 292.7 4-Cl 8.51 ± 0.02 284.5 296.0 4-Br 8.41 ± 0.02 284.0 296.8 4-NO2 7.35 ± 0.09 345.6 429.2 4-CHO 7.26 ± 0.02 309.3 346.3 3-CH3 9.32 ± 0.02 273.8 286.5 3-Phenyl 8.98 ± 0.06 282.9 304.5 3-Cl 8.00 ± 0.01 275.4 291.7 3-CHO 7.89 ± 0.04 344.6 388.4 Substituent pK1a Wavelengthb (nm) Neutral form Monoanion H 9.32 ± 0.02 274.8 288.4 4-CH3 9.47 ± 0.01 279.8 294.8 4-CH2OH 8.91 ± 0.03 279.3 292.7 4-Cl 8.51 ± 0.02 284.5 296.0 4-Br 8.41 ± 0.02 284.0 296.8 4-NO2 7.35 ± 0.09 345.6 429.2 4-CHO 7.26 ± 0.02 309.3 346.3 3-CH3 9.32 ± 0.02 273.8 286.5 3-Phenyl 8.98 ± 0.06 282.9 304.5 3-Cl 8.00 ± 0.01 275.4 291.7 3-CHO 7.89 ± 0.04 344.6 388.4 apK1 (the first pKa) was determined by UV-VIS spectral titration in 0.15 M NaCl at 25°C as described under “experimental procedures”. bThe wavelength values listed are those of the absorbance peak observed for the lowest electronic transition of the neutral and monoanionic forms of catechols. Open in new tab Table 1. Properties of C4/C3-substituted catechols. Substituent pK1a Wavelengthb (nm) Neutral form Monoanion H 9.32 ± 0.02 274.8 288.4 4-CH3 9.47 ± 0.01 279.8 294.8 4-CH2OH 8.91 ± 0.03 279.3 292.7 4-Cl 8.51 ± 0.02 284.5 296.0 4-Br 8.41 ± 0.02 284.0 296.8 4-NO2 7.35 ± 0.09 345.6 429.2 4-CHO 7.26 ± 0.02 309.3 346.3 3-CH3 9.32 ± 0.02 273.8 286.5 3-Phenyl 8.98 ± 0.06 282.9 304.5 3-Cl 8.00 ± 0.01 275.4 291.7 3-CHO 7.89 ± 0.04 344.6 388.4 Substituent pK1a Wavelengthb (nm) Neutral form Monoanion H 9.32 ± 0.02 274.8 288.4 4-CH3 9.47 ± 0.01 279.8 294.8 4-CH2OH 8.91 ± 0.03 279.3 292.7 4-Cl 8.51 ± 0.02 284.5 296.0 4-Br 8.41 ± 0.02 284.0 296.8 4-NO2 7.35 ± 0.09 345.6 429.2 4-CHO 7.26 ± 0.02 309.3 346.3 3-CH3 9.32 ± 0.02 273.8 286.5 3-Phenyl 8.98 ± 0.06 282.9 304.5 3-Cl 8.00 ± 0.01 275.4 291.7 3-CHO 7.89 ± 0.04 344.6 388.4 apK1 (the first pKa) was determined by UV-VIS spectral titration in 0.15 M NaCl at 25°C as described under “experimental procedures”. bThe wavelength values listed are those of the absorbance peak observed for the lowest electronic transition of the neutral and monoanionic forms of catechols. Open in new tab Table 2. Steady-state kinetic parameters for extradiol cleavage of substituted catechols by Mpc at 25°C.a Substituent KmA (µM) KmO2 (µM) kcat (s–1) kcat/KmA (M–1 s–1) kcat/KmO2 (M–1 s–1) pH 6.5 H 2.54 ± 0.46 10.6 ± 0.4 402 ± 7 1.58 × 108 3.79 × 107 4-CH3 1.22 ± 0.31 3.80 ± 1.8 248 ± 5 2.03 × 108 6.53 × 107 4-Cl 0.79 ± 0.52 7.11 ± 0.31 150 ± 4 1.90 × 108 2.11 × 107 4-Br 1.17 ± 0.19 10.4 ± 0.3 143 ± 3 1.22 × 108 1.38 × 107 4-CHO 15.1 ± 0.3 492 ± 73 202 ± 20 1.34 × 107 4.11 × 106 3-CHO 1.79 ± 0.22 259 ± 8 98.9 ± 3.5 5.53 × 107 3.82 × 106 pH 7.5b H 1.87 7.5 278 1.49 × 108 3.71 × 107 4-CH3 1.29 10 257 1.99 × 108 2.57 × 107 4-Cl 1.02 16 211 2.07× 108 1.32 × 107 4-CHO 22.8 313 112 4.91 × 106 3.58 × 105 3-CHO 1.97 25 58.2 2.95 × 107 2.33 × 106 3-CH3 1.64 11.0 221 1.35 × 108 2.01 × 107 3-Phenyl 0.26 190 24 9.23 × 107 1.26 × 105 Substituent KmA (µM) KmO2 (µM) kcat (s–1) kcat/KmA (M–1 s–1) kcat/KmO2 (M–1 s–1) pH 6.5 H 2.54 ± 0.46 10.6 ± 0.4 402 ± 7 1.58 × 108 3.79 × 107 4-CH3 1.22 ± 0.31 3.80 ± 1.8 248 ± 5 2.03 × 108 6.53 × 107 4-Cl 0.79 ± 0.52 7.11 ± 0.31 150 ± 4 1.90 × 108 2.11 × 107 4-Br 1.17 ± 0.19 10.4 ± 0.3 143 ± 3 1.22 × 108 1.38 × 107 4-CHO 15.1 ± 0.3 492 ± 73 202 ± 20 1.34 × 107 4.11 × 106 3-CHO 1.79 ± 0.22 259 ± 8 98.9 ± 3.5 5.53 × 107 3.82 × 106 pH 7.5b H 1.87 7.5 278 1.49 × 108 3.71 × 107 4-CH3 1.29 10 257 1.99 × 108 2.57 × 107 4-Cl 1.02 16 211 2.07× 108 1.32 × 107 4-CHO 22.8 313 112 4.91 × 106 3.58 × 105 3-CHO 1.97 25 58.2 2.95 × 107 2.33 × 106 3-CH3 1.64 11.0 221 1.35 × 108 2.01 × 107 3-Phenyl 0.26 190 24 9.23 × 107 1.26 × 105 a4-Carboxycatechol, 4-carboxymethylcatechol, and 4-tert-butylcatechol are not substrates for Mpc. 3-Chlorocatechol, 4-ethylcatechol, and 4-hydroxymethylcatechol rapidly inactivate Mpc during turnover, and making it impossible to obtain reliable kinetic parameters for these two catechols. The KmO2 value for 4-nitrocatechol is more than 700 µM; therefore, precise kinetic parameters could not be obtained for this catechol.bThe kinetic parameters for 3-formylcatechol and 3-phenylcatechol were determined in the present study; other parameters are taken from the previous study (24). Open in new tab Table 2. Steady-state kinetic parameters for extradiol cleavage of substituted catechols by Mpc at 25°C.a Substituent KmA (µM) KmO2 (µM) kcat (s–1) kcat/KmA (M–1 s–1) kcat/KmO2 (M–1 s–1) pH 6.5 H 2.54 ± 0.46 10.6 ± 0.4 402 ± 7 1.58 × 108 3.79 × 107 4-CH3 1.22 ± 0.31 3.80 ± 1.8 248 ± 5 2.03 × 108 6.53 × 107 4-Cl 0.79 ± 0.52 7.11 ± 0.31 150 ± 4 1.90 × 108 2.11 × 107 4-Br 1.17 ± 0.19 10.4 ± 0.3 143 ± 3 1.22 × 108 1.38 × 107 4-CHO 15.1 ± 0.3 492 ± 73 202 ± 20 1.34 × 107 4.11 × 106 3-CHO 1.79 ± 0.22 259 ± 8 98.9 ± 3.5 5.53 × 107 3.82 × 106 pH 7.5b H 1.87 7.5 278 1.49 × 108 3.71 × 107 4-CH3 1.29 10 257 1.99 × 108 2.57 × 107 4-Cl 1.02 16 211 2.07× 108 1.32 × 107 4-CHO 22.8 313 112 4.91 × 106 3.58 × 105 3-CHO 1.97 25 58.2 2.95 × 107 2.33 × 106 3-CH3 1.64 11.0 221 1.35 × 108 2.01 × 107 3-Phenyl 0.26 190 24 9.23 × 107 1.26 × 105 Substituent KmA (µM) KmO2 (µM) kcat (s–1) kcat/KmA (M–1 s–1) kcat/KmO2 (M–1 s–1) pH 6.5 H 2.54 ± 0.46 10.6 ± 0.4 402 ± 7 1.58 × 108 3.79 × 107 4-CH3 1.22 ± 0.31 3.80 ± 1.8 248 ± 5 2.03 × 108 6.53 × 107 4-Cl 0.79 ± 0.52 7.11 ± 0.31 150 ± 4 1.90 × 108 2.11 × 107 4-Br 1.17 ± 0.19 10.4 ± 0.3 143 ± 3 1.22 × 108 1.38 × 107 4-CHO 15.1 ± 0.3 492 ± 73 202 ± 20 1.34 × 107 4.11 × 106 3-CHO 1.79 ± 0.22 259 ± 8 98.9 ± 3.5 5.53 × 107 3.82 × 106 pH 7.5b H 1.87 7.5 278 1.49 × 108 3.71 × 107 4-CH3 1.29 10 257 1.99 × 108 2.57 × 107 4-Cl 1.02 16 211 2.07× 108 1.32 × 107 4-CHO 22.8 313 112 4.91 × 106 3.58 × 105 3-CHO 1.97 25 58.2 2.95 × 107 2.33 × 106 3-CH3 1.64 11.0 221 1.35 × 108 2.01 × 107 3-Phenyl 0.26 190 24 9.23 × 107 1.26 × 105 a4-Carboxycatechol, 4-carboxymethylcatechol, and 4-tert-butylcatechol are not substrates for Mpc. 3-Chlorocatechol, 4-ethylcatechol, and 4-hydroxymethylcatechol rapidly inactivate Mpc during turnover, and making it impossible to obtain reliable kinetic parameters for these two catechols. The KmO2 value for 4-nitrocatechol is more than 700 µM; therefore, precise kinetic parameters could not be obtained for this catechol.bThe kinetic parameters for 3-formylcatechol and 3-phenylcatechol were determined in the present study; other parameters are taken from the previous study (24). Open in new tab Table 3. Effects of pH on kcat for substituted catechols at 25°C. Substituent kcat (s–1) pH 6.5 pH 7.5 pH 8.5 H 402 278 128 4-CH3 248 257 254 4-Cl 150 211 248 4-Br 143 207 256 4-CHO 202 112 20.3a 3-CHO 98.9 58.2 37.1a Substituent kcat (s–1) pH 6.5 pH 7.5 pH 8.5 H 402 278 128 4-CH3 248 257 254 4-Cl 150 211 248 4-Br 143 207 256 4-CHO 202 112 20.3a 3-CHO 98.9 58.2 37.1a aApparent values obtained under air-saturation. Open in new tab Table 3. Effects of pH on kcat for substituted catechols at 25°C. Substituent kcat (s–1) pH 6.5 pH 7.5 pH 8.5 H 402 278 128 4-CH3 248 257 254 4-Cl 150 211 248 4-Br 143 207 256 4-CHO 202 112 20.3a 3-CHO 98.9 58.2 37.1a Substituent kcat (s–1) pH 6.5 pH 7.5 pH 8.5 H 402 278 128 4-CH3 248 257 254 4-Cl 150 211 248 4-Br 143 207 256 4-CHO 202 112 20.3a 3-CHO 98.9 58.2 37.1a aApparent values obtained under air-saturation. Open in new tab References 1. Worsey, M.J. and Williams, P.A. ( 1975 ) Metabolism of toluene and xylenes by Pseudomonas putida (arvilla) mt-2: Evidence for a new function of the TOL plasmid. J. Bacteriol. 124 , 7 –13 2. Greated, A., Lambertsen, L., Williams, P.A., and Thomas, C.M. ( 2002 ) Complete sequence of the IncP-9 TOL plasmid pWW0 from Pseudomonas putida. Environ. Microbiol. 4 , 856 –871 3. Maeda, K., Nojiri, H., Shintani, M., Yoshida, T., Habe, H., and Omori, T. 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The Journal of Biochemistry – Oxford University Press
Published: Jun 1, 2004
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