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Marked Enhancement in the Reductive Dehalogenation of Hexachloroethane by a Thr319Ala Mutation of Cytochrome P450 1A2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
249, 678–682 (1998)
RC989084
Marked Enhancement in the Reductive Dehalogenation of Hexachloroethane by a Thr319Ala Mutation of Cytochrome P450 1A2 Kazutaka Yanagita, Ikuko Sagami,1 Simon Daff, and Toru Shimizu1 Institute for Chemical Reaction Science, Tohoku University, Sendai 980-8577, Japan
Received June 26, 1998
Mutation of the conserved Thr319 residue to Ala of cytochrome P4501A2 (CYP1A2) increased the value of Vmax 9-fold for reductive dehalogenation of hexachloroethane in the reconstituted system under anaerobic conditions. The Thr319Ala mutation also increased the elimination over substitution product ratio by 5-fold. The addition of aliphatic alcohols increased by 22-fold the activity obtained with the wild type and varied the elimination over substitution product ratio. Increasing pH increased the ratio of elimination over substitution by primarily affecting the rate of elimination. q 1998 Academic Press
Cytochrome P450s (P450) are important heme enzymes responsible for the monooxygenation of drugs, hormones, lipids, etc (1-4 and references therein). The heme distal site of these enzymes is known to bind both the substrate and molecular oxygen. During catalysis, the heme and distal site residues serve to activate molecular oxygen prior to its reaction with the substrate. It has been suggested that conserved Asp/Glu or Thr residues located within the heme distal pocket are involved in this process (5-11). P450s also catalyze the reductive transformation of halogenated organic compounds (12-18). Reductive dehalogenations catalyzed by the P450 enzyme seem to be more rapid under anaerobic conditions than under aerobic conditions. Therefore, it is interesting to examine how mutations of conserved Asp/Glu and Thr residues, which are important for activating O2 during monooxygenation, to non-polar amino acids at the dis1 Correspondence authors. Fax: /81-22-217-5604, 217-5664, or 263-9849. E-mail: [email protected] or [email protected]. ac.jp. The abbreviations used are: P450, cytochrome P450; P450 1A2, rat liver microsomal cytochrome P450 1A2, CYP1A2, or cytochrome P450d; DLPC, dilauroyl-L-a-phosphatidylcholine.
tal site of P450 affect the kinetics of reductive dechlorination by the reconstituted system. In order to address the role of polar active site amino acids in catalytic dehalogenation, we have examined the effect of pH and aliphatic alcohols on the rate and product distribution of the reaction for wild type and mutant enzymes (Glu318Ala and Thr319Ala) as part of a reconstituted system, composed of purified engineered P450 1A2 (CYP1A2), NADPH-cytochrome P450 reductase and DLPC. MATERIALS AND METHODS Chemicals and reagents. Hexachloroethane and other standard compounds for product analysis were obtained from Wako Pure Chemical Industries (Osaka, Japan). Other reagents of the highest quality available were purchased from Wako Pure Chemical Industries (Osaka, Japan) and were used without further purification. Preparation of cytochrome P450 1A2. Site-directed mutagenesis, DNA sequencing, expression of wild-type and mutant CYP1A2 proteins in yeast (Saccharomyces cerevisiae), and purification of expressed CYP1A2 proteins were carried out as described previously (11, 19, 20). Purified proteins were prepared in 0.1 M potassium phosphate buffer (pH 7.4) containing 20 % (v/v) glycerol, 1 mM EDTA, and 1 mM DTT. CYP1A2 concentrations were determined using a molar absorption coefficient of 9.3 1 104 M01cm01 at 447 nm from the difference absorption spectra of the [CO-reduced] 0 [reduced] form (21, 22). Dehalogenation reaction conditions and measurements. A reaction mixture containing 1.0 mM CYP1A2, 3.0 mM cytochrome P450 reductase in buffer (0.5 mL) composed of 0.1 M potassium phosphate (pH 7.4), 20 % (v/v) glycerol, 1 mM EDTA, 1 mM NADPH, 0.5 mM DLPC and halogenated compounds was allowed to react at 25 7C. For anaerobic experiments, O2 was strictly eliminated from the solution. O2-free buffer solution and stock solutions of the substrate were made by carefully purging with 99.999% argon (Nihon Sanso Co., Tokyo) for 10 min. The reaction solution contained 75 mM glucose, 10 units of catalase, and 0.8 units of glucose oxidase for further elimination of O2 during the catalysis under an Ar atmosphere (17). Initial amounts of halogenated substrates were 0.50 mmol/0.5 mL. Stock solutions of the substrates were prepared in methanol. The final methanol concentration in each reaction solution was less than 1% (v/v), and had no significant effect on activity. After a specific time, the reaction was stopped by extracting the reaction mixture with an equal volume of diethylether three times. Aliquots (1-mL) of
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Kinetic Parameters and NADPH Oxidation Rate for Hexachloroethane Dechlorination with CYP1A2 Enzymes under Anaerobic Conditions P450 1A2
Vmaxa (mol/mol P450/min)
Kma (mM)
NADPH oxidationb (mol/mol P450/min)
Wild type Glu318Ala Thr319Ala
1.3 3.0 12.0
0.25 0.25 1.5
1.4 2.6 3.6
a Kinetic data were obtained from double reciprocal plots of the product formation velocity versus the concentration of the substrate. Experiments were repeated three times. Experimental errors were within 20%. b NADPH oxidation rates were obtained from spectral changes of NADPH absorption monitored at 340 nm in the presence of saturating concentrations of substrate during catalysis under anaerobic conditions. Experiments were repeated twice. Experimental errors were within 10%.
the 1.5-mL organic phase were analyzed by gas-chromatographymass-spectroscopy. Substrate and product concentrations were determined by comparison with authentic standards. Qualitative identification was accomplished by comparing compounds to the computerized database compiled in the mass-spectrometer. In order to ensure detection of organic acid compounds such as acetic acid, methyl ester derivatives were made with diazomethane. However, no ester derivatives were detected in this study. Qualitative experiments were carried out on a Shimadzu QP-5000 gas-chromatography-mass-spectrometer equipped a DB-64 capillary column (60 m 1 0.32 mm) (J & W Scientific, Folsom, CA). The helium carrier gas flow rate was 30 mL/min. The column temperature was held isothermally at 40 7C for 2 min and then programmed to increase to 240 7C at the rate of 5 7C/min. Detector gain was 1.5 kV. Quantitative experiments were carried out on a Shimadzu GC-8A gas-chromatography equipped a DC-550 column (3 m 1 3 mm). The nitrogen carrier gas flow rate was 60 mL/min. The column temperature was held at 90 7C. Optical absorption spectra were obtained at 25 7C with a Shimadzu UV-3100PCS spectrometer. The NADPH oxidation rate was monitored by the absorption intensity decrease at 340 nm, in the presence of saturating concentrations of substrate, that reflects the change of NADPH to NADP/.
However, it appears that the electrons supplied from NADPH to the Thr319Ala mutant during catalysis are insufficient to cause reductive dehalogenation at the rate of Vmax , implying that electrons must be acquired from another source. Table 2 gives amounts of products obtained after 20 min reaction. Tetrachloroethylene was the major product in each case, but a significant amount of pentachloroethane was also formed. Additionally, a small quantity of trichloroethylene was detected, probably resulting from the reductive dehalogenation of pentachloroethane. The far right column in Table 2 shows the ratio of elimination product over the substitution product. Under identical conditions, the wild type and mutant enzymes behaved differently as demonstrated by the overall turnover rates and by the product distribution ratios. The Glu318Ala mutation caused a 2.5-fold increase in the product formation, but retained approximately the same product distribution ratio as the wild-type enzyme. The Thr319Ala mutation caused a 3.6-fold increase in the product formation, but a 50% decrease in the rate of substitution (pentachloroethane / trichloroethylene production). This translates into a 5-fold swing in the product distribution ratio away from substitution and towards elimination. Effect of pH on the product formations. As discussed above, the Thr319Ala mutation causes a large shift in the product distribution ratio away from substitution, compared to the wild-type enzyme. Since a likely mechanistic pathway leading to substitution involves a protonation step, the effect of pH variations on the reaction rates/product distribution ratios were analyzed for both enzymes. Figs. 1A and 1B illustrate the effect on the rates of tetrachloroethylene and pentachloroethane production for the wild type and Thr319Ala mutant enzymes respectively. In both cases, lowering the pH from 8 to 6.5 resulted in decreases in the rate of tetrachloroethylene production while the rate of pentachloroethane production remained approximately constant.
RESULTS
TABLE 2
Activities for hexachloroethane in the reconstituted system. Under anaerobic conditions, the activity of wild-type CYP1A2 toward hexachloroethane was maintained for more than one hour. Both Glu318Ala and Thr319Ala mutations enhanced this activity significantly under identical conditions. Table 1 summarizes the kinetic parameters and NADPH oxidation rates, that were obtained for the wild-type and mutant enzymes under anaerobic conditions. The Thr319Ala mutation increased the value of Vmax 9-fold and increased the Km value 6-fold. For the wild type and Glu318Ala mutant, the rate of NADPH oxidation correlated broadly with the rate of substrate consumption (Table 1), suggesting a coupling of one NADPH per turnover.
Product Amounts and Relative Ratios of the Products Formed in the Catalysis with CYP1A2 Enzymes under Anaerobic Conditions Product formation P450 1A2
TetraCE
PentaCE
TriCE
TetraCE PentaCE / TriCE
Wild type Glu318Ala Thr319Ala
6.8 17 27
1.0 3.2 0.55
0.034 0.32 0.27
6.6 4.8 33
Note. Data were obtained 20 min after the reaction started. Units are expressed by nmol product content per 0.5 nmol P450 per 20 min. Experiments were repeated three times. Experimental errors were within 15%. TetraCE, tetrachloroethylene; PentaCE, pentachloroethane; TriCE, trichloroethylene.
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FIG. 1. Effects of pH on product formations from hexachloroethane with the wild type (A) and the Thr319Ala mutant (B) under anaerobic conditions at 257C. Products were tetrachloroethylene (s), pentachloroethane (n), and trichloroethylene (h). Products were monitored at 40 min after the reaction started. Experimental errors were within 20%.
Effects of alcohols on the product formations. Fig. 2 shows the effects of various alcohol concentrations on the product formation rates with the wild-type and Thr319Ala mutant enzymes. The alcohols used varied in chain-length from 1 carbon (methanol) to 4 (butanol), the magnitude of effect induced increased with alcohol concentration and with chain-length, implying that solvent polarity was the governing factor for the product formation rate. For both wild-type and mutant enzymes, the overall rate of product formation increased with the alcohol concentration by as much as 22-fold. After peaking, the rate decreased, as enzyme denaturation occurred based on the absorption band of the COferrous complex. Fig. 3 shows the changes to the product distribution ratios [tetrachloroethylene/(pentachloroethane / trichloroethylene)] for the wild type and Thr319Ala mutant on increasing alcohol concentration. These also show a pattern strongly indicative of a solvent polarity
FIG. 3. Effects of alcohols on product formation ratios [tetrachloroethylene/(pentachloroethane / trichloroethylene)] of hexachloroethane dechlorination with the wild type (A) and the Thr319Ala mutant (B) under anaerobic conditions at 257C. Product ratios were calculated from data obtained under the same conditions as in Figs. 1 and 2. Alcohols were methanol (s), ethanol (n), 1-propanol (.), and 1-butanol (h).
effect. For both enzymes, the ratio increases initially with alcohol concentration, peaking at a lower concentration than in Fig. 2, before decreasing. Therefore, reductive elimination is favored at low alcohol concentrations but substitution at higher concentrations. It is also apparent that the peaks in the product distribution ratios for the mutant occur at lower alcohol concentration than for the wild-type enzyme. The two competing effects shaping the Fig. 3 plots could both result from changes in the rate of proton supply to the enzyme’s active site. The NADPH oxidation rate obtained with the wild type enzyme in the presence of 1 M propanol was 65% of the Vmax value for dehalogenation. On the other hand, the rate obtained with the Thr319Ala mutant under the same conditions was 6.7-fold smaller than the corresponding Vmax value. Since in the absence of propanol, the rate obtained with the Thr319Ala mutant was 3.3-fold smaller than the activity (Table 1), propanol addition seems to cause the enzyme to use more electrons from non-NADPH sources. DISCUSSION
FIG. 2. Effects of alcohols on total product formations from hexachloroethane with the wild type (A) and the Thr319Ala mutant (B) under anaerobic conditions at 257C. Alcohols are methanol (s), ethanol (n), 1-propanol (.), and 1-butanol (h). Products were tetrachloroethylene, pentachloroethane, and trichloroethylene. Formation of trichloroethylene was marginal. Product formations were monitored at 40 min after the reaction started. Experimental errors were within 20%.
Table 1 shows how the rates of product formation correspond with rates for NADPH oxidation. It appears that for the wild type and mutant enzymes approximately two electrons are required for each turnover. The initial step of this reaction may be formation of a radical intermediate, on the reaction of hexachloroethane with the ferrous heme complex. A second electron could react with the radical intermediate at the heme active site to form a carbanion. The close coupling between NADPH oxidation and dechlorination with the wild type suggests that two electrons must be consumed for each turnover of the enzyme (Table 1). After carbanion formation, spontaneous chloride elimination
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and proton uptake are the two obvious pathways. Proton uptake would lead to pentachloroethane, a substitution product, whereas chloride elimination would lead to tetrachloroethane, an elimination product. Pentachloroethane probably acts as a substrate for a further reductive dechlorination leading to a trace amount of trichloroethylene. The product ratio would depend on the pathway favored for the final step. This is likely to be dependent on the environment of the carbanion, i.e. the enzyme’s active site. The dramatic shift in product distribution for the Thr319Ala mutant in favor of elimination suggests that the final step of the reactions is greatly affected. An obvious role for Thr319 in the enzyme’s active site is as a proton supply route. If this were the case, then the mutation would impede the delivery of protons and decrease the availability of protons for catalysis, clearly, elimination would be favored over substitution. In the presence of a relatively low concentration of alcohol, the product distribution ratio shifted in favor of elimination (Fig. 3). Perhaps, alcohol at the low concentration binds to the protein surface in a hydrophobic fashion and shields the heme active site from the bulk solvent water proton, resulting in a product distribution ratio in favor of elimination. As the alcohol concentration increased, the distribution ratio shifted in favor of substitution (Fig. 3). In the presence of high concentrations of alcohol, the active site of the enzyme would be expected to become more accessible to the bulk alcohol proton and the supply of protons to the active site would become faster. If proton transfer were involved in rate-determining steps in the turnover of hexachloroethane by CYP1A2 as suggested, the rate of substitution in particular would be expected to increase in the presence of excess alcohols. The most striking effect of the Thr319Ala mutation is the increase in the value of Vmax for hexachloroethane dehalogenation by 9.2-fold (Table 1). It is evident that Thr319 must be located at or near the substrate binding site of this enzyme, since the Km value with the Thr319Ala mutant was 6-fold higher than with the wild type. It is possible that a change in binding orientation of the substrate at the active site induced by the mutation could be in part the origin of the increased Vmax value. Introducing alcohol into the reaction solutions had similar effects for both wild type and mutant enzymes (Fig. 2). The major trends observed were that reactivity increased up to a peak. The sudden slowdown in activity observed at high alcohol concentration is probably due to denaturation of the enzyme. As would be expected, the peak occurs at a lower concentration of butanol and a higher concentration of methanol, consistent with size of the alkyl substituent and its hydrophobic affinity to the enzyme active site. The NADPH oxidation rate was well coupled with the Vmax value for the wild type and Glu318Ala mutant,
whereas the rate was much smaller than the Vmax value for the Thr319Ala mutant (Table 1). The NADPH oxidation rate for the wild type enzyme in the presence of alcohol was also smaller than the Vmax value. This trend was more pronounced with the Thr319Ala mutant in the presence of propanol. However, it is possible that NADPH oxidation rate for the Thr319Ala mutant is underestimated, taking consideration of the relatively high Km value of this mutant. The X-ray crystal structure of the Thr252Ala mutant of P450cam, equivalent to the Thr319Ala mutant of CYP1A2, indicates that water molecules are located at the heme distal site of the mutant, that do not exist in the wild type active site (23). Therefore, it is likely that H2O in the active site is involved in the catalysis with the Thr319Ala mutant. For both the wild type and Thr319Ala mutant enzymes, turnover was higher at higher pH (Fig. 1). It was suggested that base patches composed of Arg and Lys are located near the substrate entrance on the surface of the CYP1A2 molecule (24). If this is true, then raising pH would increase the hydrophobicity around the substrate binding site(s) and induce high affinity for the substrate. As a result, raising the pH of the enzyme solution increased only the elimination product, but not the proton-substituted product, because of the shortage of protons at the heme active site, even after the substrate enters the active site. On the other hand, the rate of formation of the substituted product (pentachloroethane) is more or less pH independent, leading to a shift in product distribution at lower pH towards substitution. It is possible therefore, that the protonation in question slows the overall rate of reaction but accelerates the substitution rate with respect to the elimination rate. Summary. The Thr319Ala mutation enormously facilitated the dehalogenation of hexachloroethane. The addition of aliphatic alcohols to the reaction solutions also largely increased the activity. The Thr319Ala mutation caused an increase in the rate of formation of the elimination product, leading to a dramatic shift in the product formation ratio. For the wild-type and mutant enzymes, the dependence of this value on pH and the large increase in rate observed in the presence of aliphatic alcohols were observed as well. ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan for Priority Area (biometallics) (10129201) to T.S.
REFERENCES 1. Guengerich, F. P. (1991) J. Biol. Chem. 266, 10019–10022. 2. Johnson, E. F., and Waterman, M. R., Eds. (1996) Methods in Enzymology Vol. 272, Academic Press, San Diego.
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3. Ortiz de Montellano, P. R., Ed. (1995) Cytochrome P450, Structure, Mechanism and Biochemistry, 2nd Ed., Plenum Press, New York. 4. Porter, T. D., and Coon, M. J. (1991) J. Biol. Chem. 266, 13469– 13472. 5. Raag, R., Martinis, S. A., Sligar, S. G., and Poulos, T. L. (1991) Biochemistry 26, 8165–8174. 6. Imai, M., Shimada, H., Watanabe, Y., Matsushima-Hibiya, Y., Makino, R., Koga, H., Horiuchi, T., and Ishimura, Y. (1989) Proc. Natl. Acad. Sci. USA 86, 7823–7827. 7. Martinis, A. S., Atkins, W. M., Stayton, P. S., and Sligar, S. G. (1989) J. Am. Chem. Soc. 111, 9252–9253. 8. Yeom, H., Sligar, S. G., Li, H., Poulos, T. L., and Fulco, A. J. (1995) Biochemistry 34, 14733–14740. 9. Gerber, N. C., and Sligar, S. G. (1992) J. Am. Chem. Soc. 114, 8742–8743. 10. Gerber, N. C., and Sligar, S. G. (1994) J. Biol. Chem. 269, 4260– 4266. 11. Ishigooka, M., Shimizu, T., Hiroya, K., and Hatano, M. (1992) Biochemistry 31, 1528–1531. 12. Goeptar, A. R., Scheerens, H., and Vermeulen, N. P. E. (1995) Crit. Rev. Toxicol. 25, 25–65. 13. Baker, M. T., Vasquez, M. T., and Chiang, C.-K. (1991) Biochem. Pharmacol. 41, 1691–1699.
14. Castro, C. E., Wade, R. S., and Belser, N. O. (1985) Biochemistry 24, 204–210. 15. Miller, R. E., and Guengerich, F. P. (1982) Biochemistry 21, 1090–1097. 16. Yanagita, K., Sagami, I., and Shimizu, T. (1997) Chemistry Lett. 561–562. 17. Yanagita, K., Sagami, I., and Shimizu, T. (1997) Arch. Biochem. Biophys. 346, 269–276. 18. Wackett, L. P. (1995) in Microbial Transformation and Degradation of Toxic Organic Chemicals (Young, L. Y., and Cerniglia, C. E., Eds.), pp. 217–241, Wiley-Liss, New York. 19. Shimizu, T., Murakami, Y., and Hatano, M. (1994) J. Biol. Chem. 269, 13296–13304. 20. Nakano, R., Sato, H., Watanabe, A., Ito, O., and Shimizu, T. (1996) J. Biol. Chem. 271, 8570–8574. 21. Shimizu, T., Sadeque, A. J. M., Sadeque, G. N., Hatano, M., and Fujii-Kuriyama, Y. (1991) Biochemistry 30, 1490–1496. 22. Shimizu, T., Ito, O., Hatano, M., and Fujii-Kuriyama, Y. (1991) Biochemistry 30, 4659–4662. 23. Raag, R., Martinis, S. A., Sligar, S. G., and Poulos, T. L. (1991) Biochemistry 30, 11420–11429. 24. Krainev, A. G., Shimizu, T., Hiroya, K., and Hatano, M. (1992) Arch. Biochem. Biophys. 298, 198–203.
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249, 678–682 (1998)
RC989084
Marked Enhancement in the Reductive Dehalogenation of Hexachloroethane by a Thr319Ala Mutation of Cytochrome P450 1A2 Kazutaka Yanagita, Ikuko Sagami,1 Simon Daff, and Toru Shimizu1 Institute for Chemical Reaction Science, Tohoku University, Sendai 980-8577, Japan
Received June 26, 1998
Mutation of the conserved Thr319 residue to Ala of cytochrome P4501A2 (CYP1A2) increased the value of Vmax 9-fold for reductive dehalogenation of hexachloroethane in the reconstituted system under anaerobic conditions. The Thr319Ala mutation also increased the elimination over substitution product ratio by 5-fold. The addition of aliphatic alcohols increased by 22-fold the activity obtained with the wild type and varied the elimination over substitution product ratio. Increasing pH increased the ratio of elimination over substitution by primarily affecting the rate of elimination. q 1998 Academic Press
Cytochrome P450s (P450) are important heme enzymes responsible for the monooxygenation of drugs, hormones, lipids, etc (1-4 and references therein). The heme distal site of these enzymes is known to bind both the substrate and molecular oxygen. During catalysis, the heme and distal site residues serve to activate molecular oxygen prior to its reaction with the substrate. It has been suggested that conserved Asp/Glu or Thr residues located within the heme distal pocket are involved in this process (5-11). P450s also catalyze the reductive transformation of halogenated organic compounds (12-18). Reductive dehalogenations catalyzed by the P450 enzyme seem to be more rapid under anaerobic conditions than under aerobic conditions. Therefore, it is interesting to examine how mutations of conserved Asp/Glu and Thr residues, which are important for activating O2 during monooxygenation, to non-polar amino acids at the dis1 Correspondence authors. Fax: /81-22-217-5604, 217-5664, or 263-9849. E-mail: [email protected] or [email protected]. ac.jp. The abbreviations used are: P450, cytochrome P450; P450 1A2, rat liver microsomal cytochrome P450 1A2, CYP1A2, or cytochrome P450d; DLPC, dilauroyl-L-a-phosphatidylcholine.
tal site of P450 affect the kinetics of reductive dechlorination by the reconstituted system. In order to address the role of polar active site amino acids in catalytic dehalogenation, we have examined the effect of pH and aliphatic alcohols on the rate and product distribution of the reaction for wild type and mutant enzymes (Glu318Ala and Thr319Ala) as part of a reconstituted system, composed of purified engineered P450 1A2 (CYP1A2), NADPH-cytochrome P450 reductase and DLPC. MATERIALS AND METHODS Chemicals and reagents. Hexachloroethane and other standard compounds for product analysis were obtained from Wako Pure Chemical Industries (Osaka, Japan). Other reagents of the highest quality available were purchased from Wako Pure Chemical Industries (Osaka, Japan) and were used without further purification. Preparation of cytochrome P450 1A2. Site-directed mutagenesis, DNA sequencing, expression of wild-type and mutant CYP1A2 proteins in yeast (Saccharomyces cerevisiae), and purification of expressed CYP1A2 proteins were carried out as described previously (11, 19, 20). Purified proteins were prepared in 0.1 M potassium phosphate buffer (pH 7.4) containing 20 % (v/v) glycerol, 1 mM EDTA, and 1 mM DTT. CYP1A2 concentrations were determined using a molar absorption coefficient of 9.3 1 104 M01cm01 at 447 nm from the difference absorption spectra of the [CO-reduced] 0 [reduced] form (21, 22). Dehalogenation reaction conditions and measurements. A reaction mixture containing 1.0 mM CYP1A2, 3.0 mM cytochrome P450 reductase in buffer (0.5 mL) composed of 0.1 M potassium phosphate (pH 7.4), 20 % (v/v) glycerol, 1 mM EDTA, 1 mM NADPH, 0.5 mM DLPC and halogenated compounds was allowed to react at 25 7C. For anaerobic experiments, O2 was strictly eliminated from the solution. O2-free buffer solution and stock solutions of the substrate were made by carefully purging with 99.999% argon (Nihon Sanso Co., Tokyo) for 10 min. The reaction solution contained 75 mM glucose, 10 units of catalase, and 0.8 units of glucose oxidase for further elimination of O2 during the catalysis under an Ar atmosphere (17). Initial amounts of halogenated substrates were 0.50 mmol/0.5 mL. Stock solutions of the substrates were prepared in methanol. The final methanol concentration in each reaction solution was less than 1% (v/v), and had no significant effect on activity. After a specific time, the reaction was stopped by extracting the reaction mixture with an equal volume of diethylether three times. Aliquots (1-mL) of
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Kinetic Parameters and NADPH Oxidation Rate for Hexachloroethane Dechlorination with CYP1A2 Enzymes under Anaerobic Conditions P450 1A2
Vmaxa (mol/mol P450/min)
Kma (mM)
NADPH oxidationb (mol/mol P450/min)
Wild type Glu318Ala Thr319Ala
1.3 3.0 12.0
0.25 0.25 1.5
1.4 2.6 3.6
a Kinetic data were obtained from double reciprocal plots of the product formation velocity versus the concentration of the substrate. Experiments were repeated three times. Experimental errors were within 20%. b NADPH oxidation rates were obtained from spectral changes of NADPH absorption monitored at 340 nm in the presence of saturating concentrations of substrate during catalysis under anaerobic conditions. Experiments were repeated twice. Experimental errors were within 10%.
the 1.5-mL organic phase were analyzed by gas-chromatographymass-spectroscopy. Substrate and product concentrations were determined by comparison with authentic standards. Qualitative identification was accomplished by comparing compounds to the computerized database compiled in the mass-spectrometer. In order to ensure detection of organic acid compounds such as acetic acid, methyl ester derivatives were made with diazomethane. However, no ester derivatives were detected in this study. Qualitative experiments were carried out on a Shimadzu QP-5000 gas-chromatography-mass-spectrometer equipped a DB-64 capillary column (60 m 1 0.32 mm) (J & W Scientific, Folsom, CA). The helium carrier gas flow rate was 30 mL/min. The column temperature was held isothermally at 40 7C for 2 min and then programmed to increase to 240 7C at the rate of 5 7C/min. Detector gain was 1.5 kV. Quantitative experiments were carried out on a Shimadzu GC-8A gas-chromatography equipped a DC-550 column (3 m 1 3 mm). The nitrogen carrier gas flow rate was 60 mL/min. The column temperature was held at 90 7C. Optical absorption spectra were obtained at 25 7C with a Shimadzu UV-3100PCS spectrometer. The NADPH oxidation rate was monitored by the absorption intensity decrease at 340 nm, in the presence of saturating concentrations of substrate, that reflects the change of NADPH to NADP/.
However, it appears that the electrons supplied from NADPH to the Thr319Ala mutant during catalysis are insufficient to cause reductive dehalogenation at the rate of Vmax , implying that electrons must be acquired from another source. Table 2 gives amounts of products obtained after 20 min reaction. Tetrachloroethylene was the major product in each case, but a significant amount of pentachloroethane was also formed. Additionally, a small quantity of trichloroethylene was detected, probably resulting from the reductive dehalogenation of pentachloroethane. The far right column in Table 2 shows the ratio of elimination product over the substitution product. Under identical conditions, the wild type and mutant enzymes behaved differently as demonstrated by the overall turnover rates and by the product distribution ratios. The Glu318Ala mutation caused a 2.5-fold increase in the product formation, but retained approximately the same product distribution ratio as the wild-type enzyme. The Thr319Ala mutation caused a 3.6-fold increase in the product formation, but a 50% decrease in the rate of substitution (pentachloroethane / trichloroethylene production). This translates into a 5-fold swing in the product distribution ratio away from substitution and towards elimination. Effect of pH on the product formations. As discussed above, the Thr319Ala mutation causes a large shift in the product distribution ratio away from substitution, compared to the wild-type enzyme. Since a likely mechanistic pathway leading to substitution involves a protonation step, the effect of pH variations on the reaction rates/product distribution ratios were analyzed for both enzymes. Figs. 1A and 1B illustrate the effect on the rates of tetrachloroethylene and pentachloroethane production for the wild type and Thr319Ala mutant enzymes respectively. In both cases, lowering the pH from 8 to 6.5 resulted in decreases in the rate of tetrachloroethylene production while the rate of pentachloroethane production remained approximately constant.
RESULTS
TABLE 2
Activities for hexachloroethane in the reconstituted system. Under anaerobic conditions, the activity of wild-type CYP1A2 toward hexachloroethane was maintained for more than one hour. Both Glu318Ala and Thr319Ala mutations enhanced this activity significantly under identical conditions. Table 1 summarizes the kinetic parameters and NADPH oxidation rates, that were obtained for the wild-type and mutant enzymes under anaerobic conditions. The Thr319Ala mutation increased the value of Vmax 9-fold and increased the Km value 6-fold. For the wild type and Glu318Ala mutant, the rate of NADPH oxidation correlated broadly with the rate of substrate consumption (Table 1), suggesting a coupling of one NADPH per turnover.
Product Amounts and Relative Ratios of the Products Formed in the Catalysis with CYP1A2 Enzymes under Anaerobic Conditions Product formation P450 1A2
TetraCE
PentaCE
TriCE
TetraCE PentaCE / TriCE
Wild type Glu318Ala Thr319Ala
6.8 17 27
1.0 3.2 0.55
0.034 0.32 0.27
6.6 4.8 33
Note. Data were obtained 20 min after the reaction started. Units are expressed by nmol product content per 0.5 nmol P450 per 20 min. Experiments were repeated three times. Experimental errors were within 15%. TetraCE, tetrachloroethylene; PentaCE, pentachloroethane; TriCE, trichloroethylene.
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FIG. 1. Effects of pH on product formations from hexachloroethane with the wild type (A) and the Thr319Ala mutant (B) under anaerobic conditions at 257C. Products were tetrachloroethylene (s), pentachloroethane (n), and trichloroethylene (h). Products were monitored at 40 min after the reaction started. Experimental errors were within 20%.
Effects of alcohols on the product formations. Fig. 2 shows the effects of various alcohol concentrations on the product formation rates with the wild-type and Thr319Ala mutant enzymes. The alcohols used varied in chain-length from 1 carbon (methanol) to 4 (butanol), the magnitude of effect induced increased with alcohol concentration and with chain-length, implying that solvent polarity was the governing factor for the product formation rate. For both wild-type and mutant enzymes, the overall rate of product formation increased with the alcohol concentration by as much as 22-fold. After peaking, the rate decreased, as enzyme denaturation occurred based on the absorption band of the COferrous complex. Fig. 3 shows the changes to the product distribution ratios [tetrachloroethylene/(pentachloroethane / trichloroethylene)] for the wild type and Thr319Ala mutant on increasing alcohol concentration. These also show a pattern strongly indicative of a solvent polarity
FIG. 3. Effects of alcohols on product formation ratios [tetrachloroethylene/(pentachloroethane / trichloroethylene)] of hexachloroethane dechlorination with the wild type (A) and the Thr319Ala mutant (B) under anaerobic conditions at 257C. Product ratios were calculated from data obtained under the same conditions as in Figs. 1 and 2. Alcohols were methanol (s), ethanol (n), 1-propanol (.), and 1-butanol (h).
effect. For both enzymes, the ratio increases initially with alcohol concentration, peaking at a lower concentration than in Fig. 2, before decreasing. Therefore, reductive elimination is favored at low alcohol concentrations but substitution at higher concentrations. It is also apparent that the peaks in the product distribution ratios for the mutant occur at lower alcohol concentration than for the wild-type enzyme. The two competing effects shaping the Fig. 3 plots could both result from changes in the rate of proton supply to the enzyme’s active site. The NADPH oxidation rate obtained with the wild type enzyme in the presence of 1 M propanol was 65% of the Vmax value for dehalogenation. On the other hand, the rate obtained with the Thr319Ala mutant under the same conditions was 6.7-fold smaller than the corresponding Vmax value. Since in the absence of propanol, the rate obtained with the Thr319Ala mutant was 3.3-fold smaller than the activity (Table 1), propanol addition seems to cause the enzyme to use more electrons from non-NADPH sources. DISCUSSION
FIG. 2. Effects of alcohols on total product formations from hexachloroethane with the wild type (A) and the Thr319Ala mutant (B) under anaerobic conditions at 257C. Alcohols are methanol (s), ethanol (n), 1-propanol (.), and 1-butanol (h). Products were tetrachloroethylene, pentachloroethane, and trichloroethylene. Formation of trichloroethylene was marginal. Product formations were monitored at 40 min after the reaction started. Experimental errors were within 20%.
Table 1 shows how the rates of product formation correspond with rates for NADPH oxidation. It appears that for the wild type and mutant enzymes approximately two electrons are required for each turnover. The initial step of this reaction may be formation of a radical intermediate, on the reaction of hexachloroethane with the ferrous heme complex. A second electron could react with the radical intermediate at the heme active site to form a carbanion. The close coupling between NADPH oxidation and dechlorination with the wild type suggests that two electrons must be consumed for each turnover of the enzyme (Table 1). After carbanion formation, spontaneous chloride elimination
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and proton uptake are the two obvious pathways. Proton uptake would lead to pentachloroethane, a substitution product, whereas chloride elimination would lead to tetrachloroethane, an elimination product. Pentachloroethane probably acts as a substrate for a further reductive dechlorination leading to a trace amount of trichloroethylene. The product ratio would depend on the pathway favored for the final step. This is likely to be dependent on the environment of the carbanion, i.e. the enzyme’s active site. The dramatic shift in product distribution for the Thr319Ala mutant in favor of elimination suggests that the final step of the reactions is greatly affected. An obvious role for Thr319 in the enzyme’s active site is as a proton supply route. If this were the case, then the mutation would impede the delivery of protons and decrease the availability of protons for catalysis, clearly, elimination would be favored over substitution. In the presence of a relatively low concentration of alcohol, the product distribution ratio shifted in favor of elimination (Fig. 3). Perhaps, alcohol at the low concentration binds to the protein surface in a hydrophobic fashion and shields the heme active site from the bulk solvent water proton, resulting in a product distribution ratio in favor of elimination. As the alcohol concentration increased, the distribution ratio shifted in favor of substitution (Fig. 3). In the presence of high concentrations of alcohol, the active site of the enzyme would be expected to become more accessible to the bulk alcohol proton and the supply of protons to the active site would become faster. If proton transfer were involved in rate-determining steps in the turnover of hexachloroethane by CYP1A2 as suggested, the rate of substitution in particular would be expected to increase in the presence of excess alcohols. The most striking effect of the Thr319Ala mutation is the increase in the value of Vmax for hexachloroethane dehalogenation by 9.2-fold (Table 1). It is evident that Thr319 must be located at or near the substrate binding site of this enzyme, since the Km value with the Thr319Ala mutant was 6-fold higher than with the wild type. It is possible that a change in binding orientation of the substrate at the active site induced by the mutation could be in part the origin of the increased Vmax value. Introducing alcohol into the reaction solutions had similar effects for both wild type and mutant enzymes (Fig. 2). The major trends observed were that reactivity increased up to a peak. The sudden slowdown in activity observed at high alcohol concentration is probably due to denaturation of the enzyme. As would be expected, the peak occurs at a lower concentration of butanol and a higher concentration of methanol, consistent with size of the alkyl substituent and its hydrophobic affinity to the enzyme active site. The NADPH oxidation rate was well coupled with the Vmax value for the wild type and Glu318Ala mutant,
whereas the rate was much smaller than the Vmax value for the Thr319Ala mutant (Table 1). The NADPH oxidation rate for the wild type enzyme in the presence of alcohol was also smaller than the Vmax value. This trend was more pronounced with the Thr319Ala mutant in the presence of propanol. However, it is possible that NADPH oxidation rate for the Thr319Ala mutant is underestimated, taking consideration of the relatively high Km value of this mutant. The X-ray crystal structure of the Thr252Ala mutant of P450cam, equivalent to the Thr319Ala mutant of CYP1A2, indicates that water molecules are located at the heme distal site of the mutant, that do not exist in the wild type active site (23). Therefore, it is likely that H2O in the active site is involved in the catalysis with the Thr319Ala mutant. For both the wild type and Thr319Ala mutant enzymes, turnover was higher at higher pH (Fig. 1). It was suggested that base patches composed of Arg and Lys are located near the substrate entrance on the surface of the CYP1A2 molecule (24). If this is true, then raising pH would increase the hydrophobicity around the substrate binding site(s) and induce high affinity for the substrate. As a result, raising the pH of the enzyme solution increased only the elimination product, but not the proton-substituted product, because of the shortage of protons at the heme active site, even after the substrate enters the active site. On the other hand, the rate of formation of the substituted product (pentachloroethane) is more or less pH independent, leading to a shift in product distribution at lower pH towards substitution. It is possible therefore, that the protonation in question slows the overall rate of reaction but accelerates the substitution rate with respect to the elimination rate. Summary. The Thr319Ala mutation enormously facilitated the dehalogenation of hexachloroethane. The addition of aliphatic alcohols to the reaction solutions also largely increased the activity. The Thr319Ala mutation caused an increase in the rate of formation of the elimination product, leading to a dramatic shift in the product formation ratio. For the wild-type and mutant enzymes, the dependence of this value on pH and the large increase in rate observed in the presence of aliphatic alcohols were observed as well. ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan for Priority Area (biometallics) (10129201) to T.S.
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