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Chem. Res. Toxicol. 2003, 16, 668-675

Catechol Estrogen 4-Hydroxyequilenin Is a Substrate and an Inhibitor of Catechol-O-Methyltransferase Jiaqin Yao,† Yan Li,† Minsun Chang,† Huaping Wu,† Xiaofeng Yang,† Julie E. Goodman,‡,§ Xuemei Liu,† Hong Liu,† Andrew D. Mesecar,| Richard B. van Breemen,† James D. Yager,‡ and Judy L. Bolton*,† Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612, Division of Toxicological Sciences, Department of Environmental Health Sciences, Johns Hopkins University School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205, and Center for Pharmaceutical Biotechnology (M/C 870), College of Pharmacy, University of Illinois at Chicago, 900 S. Ashland Avenue, Chicago, Illinois 60607 Received March 19, 2003

Redox and/or electrophilic metabolites formed during estrogen metabolism may play a role in estrogen carcinogenesis. 4-Hydroxyequilenin (4-OHEN) is the major phase I catechol metabolite of the equine estrogens equilenin and equilin, which are components of the most widely prescribed estrogen replacement formulation, Premarin. Previously, we have found that 4-OHEN rapidly autoxidized to an o-quinone in vitro and caused toxic effects such as the inactivation of human detoxification enzymes. 4-OHEN has also been shown to be a substrate for catechol-O-methyltransferase (COMT) in human breast cancer cells. In the present study, we demonstrated that 4-OHEN was not only a substrate of recombinant human soluble COMT in vitro with a Km of 2.4 µM and kcat of 6.0 min-1 but it also inhibited its own methylation by COMT at higher concentrations in the presence of the reducing agent dithiothreitol. In addition, 4-OHEN was found to be an irreversible inhibitor of COMT-catalyzed methylation of the endogenous catechol estrogen 4-hydroxyestradiol with a Ki of 26.0 µM and a k2 of 1.62 × 10-2 s-1. 4-OHEN in vitro not only caused the formation of intermolecular disulfide bonds as demonstrated by gel electrophoresis, but electrospray ionization mass spectrometry and matrixassisted laser desorption ionization time-of-flight mass spectrometry also showed that 4-OHEN alkylated multiple residues of COMT. Peptide mapping experiments further indicated that Cys33 in recombinant human soluble COMT was the residue most likely modified by 4-OHEN in vitro. These data suggest that inhibition of COMT methylation by 4-OHEN might reduce endogenous catechol estrogen clearance in vivo and further enhance toxicity.

Introduction COMT1 (EC 2.1.1.6) catalyzes the transfer of a methyl group from the donor SAM to a catechol substrate (1). Catecholamine neurotransmitters and endogenous catechol estrogens are the most important physiological substrates of COMT (2, 3). COMT also plays an important role in the inactivation of biologically active and toxic catechols (1). In mammals, COMT is widely distributed in brain and peripheral tissues (4). There are two * To whom correspondence should be addressed. Tel: (312)996-5280. Fax: (312)996-7107. E-mail: [email protected]. † Department of Medicinal Chemistry and Pharmacognosy (M/C 781), University of Illinois at Chicago. ‡ Johns Hopkins University School of Hygiene and Public Health. § Present address: National Cancer Institute, NIH, 6130 Executive Blvd., T-41, Bethesda, Maryland 20892. | Center for Pharmaceutical Biotechnology (M/C 870), University of Illinois at Chicago. 1 Abbreviations: CHCA, R-cyano-4-hydroxycinnamic acid; COMT, catechol-O-methyltransferase; DTT, dithiothreitol; ESI-MS, electrospray ionization mass spectrometry; GST, glutathione S-transferase; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-offlight mass spectrometry; MB-COMT, membrane-bound catechol-Omethyltransferase; 2-MeOE2, 2-methoxyestradiol; 4-MeOE2, 4-methoxyestradiol; 4-MeOEN, 4-methoxyequilenin; 2-OHE2, 2-hydroxyestradiol; 4-OHE2, 4-hydroxyestradiol; 4-OHEN, 4-hydroxyequilenin; PCBs, polychlorinated biphenyls; SAM, S-adenosyl-L-methionine; S-COMT, soluble catechol-O-methyltransferase.

isoforms of COMT, soluble (S-COMT) and membranebound (MB-COMT), which are encoded by a single gene with different transcription start sites. S-COMT is the predominantly expressed isoform in most human tissues (5). Both S-COMT and MB-COMT have wild-type (Val108 in S-COMT and Val158 in MB-COMT) and variant (Met108 in S-COMT and Met158 in MB-COMT) forms, and the variant forms have 3-4-fold lower enzyme activity as compared to the wild-type forms with some substrates (6). Several, but not all, epidemiological studies have shown that women homozygous with the variant COMT form have an increased risk of developing breast cancer (7-10), which may be related to their decreased ability to detoxify catechol estrogen metabolites. Recent clinical studies have shown that hormone replacement therapy is associated with an increased risk of developing breast cancer (11). Although the molecular mechanism(s) for estrogen carcinogenesis are still unclear, quinones formed during estrogen metabolism may cause redox cycling or act as electrophiles and contribute to mutation and tumor formation. 4-OHEN is the major phase I catechol metabolite of the equine estrogens equilenin and equilin that make up approximately 50% of the most widely prescribed estrogen replacement formulation, Premarin (12, 13). 4-OHEN autoxidizes to

10.1021/tx0340549 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/23/2003

4-OHEN Is a Substrate and Inhibitor of COMT

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Scheme 1. Proposed Mechanisms for Inactivation of Human S-COMT Mediated by 4-OHENa

a

[H] refers to any reducing agent, and n is the number of Cys residues in COMT.

an o-quinone (Scheme 1), which induces a variety of DNA lesions in vitro including depurination, formation of bulky stable adducts, and oxidation of the phosphate sugar backbone and purine/pyrimidine bases (12-16). In human breast cancer cells, 4-OHEN was found to induce DNA damage and apoptosis (17), and DNA damage was also observed in vivo in the mammary tissues of rats treated with 4-OHEN (18). In addition, 4-OHEN has the potential to be an effective tumor promoter and complete carcinogen in vitro (19). Besides its genotoxic effects, 4-OHEN has also been shown to inactivate human detoxification enzymes in vitro and in cultured human cells (20-22). Recent studies indicated that the catechol metabolite 4-OHEN was an intermediate in the metabolism of equilenin and was methylated by COMT in human breast cancer cells (MCF-7 and MDA-MB-231) producing 4-MeOEN (Scheme 1, 23). Because it has been shown in vitro that 4-OHEN rapidly autoxidized to an o-quinone (24), it was of interest to examine in vitro whether methylation of 4-OHEN by COMT occurs and whether 4-OHEN could inhibit the methylation activity of COMT. In the present investigation, changes in the methylation activity of recombinant human wild-type S-COMT were studied in the presence of 4-OHEN. The data showed that 4-OHEN was not only a substrate of COMT but also inhibited the COMTcatalyzed methylation of 4-OHE2. 4-OHEN caused oxidative damage to COMT resulting in the formation of intermolecular disulfide bonds. Finally, ESI-MS and MALDI-TOF-MS showed that 4-OHEN could covalently modify multiple residues of COMT in vitro.

Materials and Methods Caution: The catechol estrogens were handled in accordance with NIH guidelines for the Laboratory Use of Chemical Carcinogens (25).

Materials. All chemicals were purchased from Sigma (St. Louis, MO), Aldrich (Milwaukee, WI), or Fisher Scientific (Itasca, IL) unless stated otherwise. 2-MeOE2 was purchased from Steraloids (Newport, RI). 4-OHEN was synthesized by treating equilin with Fremy’s salt as described previously (26, 27) with minor modifications (24). 4-MeOEN was synthesized as described previously (23, 28) and used as the standard to quantitate the product after incubation of 4-OHEN with COMT. Recombinant human wild-type S-COMT was prepared as described previously (29) and stored in PBS with 7% glycerol and 1 mM DTT at -80 °C. Enzyme Assays and Kinetic Measurements. COMT activity was determined by HPLC as described previously (29, 30) with minor modifications. The reaction mixture (240 µL) containing 200 mM sodium phosphate buffer (pH 7.8), 5 mM MgCl2, 1 mM DTT, 0.2 mM SAM, and 50 pmol COMT was preincubated for 3 min at 37 °C. The reaction was initiated by adding 10 µL of various concentrations of 4-OHEN in DMSO and terminated after 3 min by addition of 25 µL of 4 M perchloric acid. Following centrifugation to precipitate COMT, the supernatant (245 µL) was mixed with 5 µL of 125 µM 2-MeOE2 (internal standard; final concentration, 2.5 µM), and 200 µL of each sample was injected for HPLC analysis. When 4-OHE2 (100 µM) was used as the substrate to measure COMT activity, the final reaction volume was 500 µL and the concentration of the other reagents was as described above. Kinetic parameters (Km and kcat) were determined by fitting velocity vs concentration data to the Michaelis-Menten equation using the nonlinear regression analysis program in SigmaPlot (SPSS, Chicago, IL). HPLC Conditions. HPLC analyses were performed using a 4.6 mm × 250 mm ultrasphere C18 column (Beckman, Fullerton, CA) on a Shimadzu LC-10A gradient HPLC equipped with a SIL-10A auto injector, SPD-M10AV UV/vis photodiode array detector (280 nm), and SPD-10AV detector (Columbia, MD). The mobile phase consisted of 40% methanol in 0.5% perchloric acid/0.5% acetic acid (pH 3.5) at a flow rate of 1.0 mL/min for 5 min, increased to 65% methanol over the next 45 min, and increased to 90% methanol in 5 min.

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Inhibition of COMT Activity by 4-OHEN In Vitro. COMT was reduced with 10 mM DTT at 37 °C for 30 min and then passed through a NAP-5 column (Amersham Pharmacia Biotech, Piscataway, NJ) to remove DTT. COMT (20 µM) was preincubated at 37 °C in 200 mM potassium phosphate buffer (pH 7.8) with various concentrations of 4-OHEN or DMSO, in the presence or absence of 1 mM DTT. In some samples where DTT had been removed, 1 mM DTT was added after a 10 min incubation with 4-OHEN. Aliquots (5 µL) were removed at various times and diluted 100-fold into the assay buffer in a total volume of 500 µL. COMT activity was measured using 4-OHE2 as the substrate as described above. The inhibition of COMT methylation activity by 4-OHEN was compared to the DMSO control, and inhibitory activity was expressed by percent of control enzyme activity. Gel filtration experiments with 4-OHEN-treated COMT were performed using NAP-5 columns. Inhibition kinetic studies were performed with 2-50 µM 4-OHEN according to Kitz and Wilson (31). Electrophoretic Analyses of COMT Treated with 4-OHEN. COMT modified by 4-OHEN was analyzed using nonreducing SDS-PAGE. Briefly, COMT (20 µM) without DTT was incubated with various concentrations of 4-OHEN or DMSO in 50 mM ammonium bicarbonate buffer (pH 8.0) at 37 °C for 15 min. In some cases, 10 mM DTT was further incubated with 4-OHEN-treated COMT for an additional 15 min. Treated samples (50 µL) were mixed with 10 µL of sample buffer without β-mercaptoethanol, followed by incubation at 100 °C for 5 min, and then subjected to SDS-PAGE. The protein bands were visualized after staining with Coomassie Brilliant Blue R250. ESI-MS. For analysis of COMT covalently modified by 4-OHEN, 20 µM COMT in 50 mM ammonium bicarbonate (pH 8.0) was incubated with various concentrations of 4-OHEN for 15 min at room temperature. The samples were diluted with 50% methanol/0.2% formic acid to give a final concentration of 1 µM, infused into the ion source at 20 µL/min using a syringe pump, and analyzed by ESI-MS using a Micromass Quattro II triple quadrupole mass spectrometer (Manchester, U.K.). Only positive ion electrospray mass spectra were recorded. Nitrogen was used as the drying and nebulizing gas at 8 and 0.8 L/min, respectively. Typical operating parameters were as follows: capillary voltage, 3.5 kV; source temperature, 120 °C; and cone voltage, 35 V. The spectra were scanned over the range of m/z 800-2000 at 6 s/scan, and the run duration was set to 2 min. The deconvoluted spectra were obtained using MaxEnt 1 software (Micromass Co.). MALDI-TOF-MS. 4-OHEN-modified COMT was also analyzed by MALDI-TOF-MS. COMT (20 µM) in 50 mM ammonium bicarbonate (pH 8.0) was incubated with various concentrations of 4-OHEN for 15 min at room temperature, and aliquots were combined with 5-fold volumes of matrix solution (saturated sinapinic acid in 30% acetonitrile containing 0.3% trifluoroacetic acid). Samples (0.5 µL) were spotted on the MALDI target and air-dried immediately prior to analysis. MALDI-TOF mass spectra were obtained using a Voyager-DE Pro instrument (Applied Biosystems, Foster City, CA) equipped with a nitrogen laser in the positive-ion mode. A total of 200 mass spectra were acquired and signal-averaged per analysis. COMT was analyzed in the linear TOF mode using an accelerating voltage of 20 kV, and peptide digests of COMT were analyzed in reflectron mode at 20 kV. Peptide Mapping of COMT Modified by 4-OHEN. COMT (20 µM) in 50 mM ammonium bicarbonate (pH 8.0) was incubated with various concentrations of 4-OHEN or DMSO for 15 min at room temperature in a total volume of 50 µL. For CNBr cleavage, 4-OHEN-treated COMT was acidified by the addition of 5 µL of 1.0 M HCl and then incubated at room temperature in the dark for 24 h after adding CNBr (∼25 µg). The mixture was then dried under a stream of nitrogen gas and redissolved in water containing 0.1% TFA. The CNBr-cleaved peptides were purified by using a ZipTipC18 (Millipore, Bedford, MA) according to the protocol provided by the manufacturer and then mixed in a 1:1 ratio with saturated CHCA solution in 50:

Yao et al.

Figure 1. HPLC analysis of 4-OHEN incubated with COMT. 4-OHEN (2 µM) was incubated with COMT (200 nM) in reaction buffer for 3 min (A) in the absence of 1 mM DTT, (B) in the presence of 1 mM DTT, or (C) in the presence of 1 mM NADPH. After addition of 25 µL of perchloric acid, 2-MeOE2 (2.5 µM) was added as an internal standard and the sample was analyzed by HPLC as described in the Materials and Methods. 50 acetonitrile/water containing 0.1% TFA. Each sample (0.5 µL) was applied to the sample target for MALDI-TOF-MS analysis. For trypsin digestion, treated COMT was mixed with a 1/10 volume of 250 mM NH4HCO3, and the pH was adjusted to 8.0 with aqueous ammonia. A trypsin solution (2 mg/mL) was added at the weight ratio of 1:50 trypsin/protein. The mixture was incubated at 37 °C for 24 h and prepared as described above for MALDI-TOF-MS analysis.

Results Methylation of 4-OHEN. Previously, we showed that the equine catechol estrogen 4-OHEN rapidly autoxidized to 4-OHEN-o-quinone (Scheme 1, 24). In addition, we have also detected 4-MeOEN as a metabolite of equilenin in breast cancer cell lines (23). In the present study, it was of interest to determine if 4-OHEN was a substrate and/or potential inhibitor of recombinant human soluble COMT. As seen in Figure 1A, no 4-MeOEN was detected in the absence of the reducing agent DTT. However, in the presence of DTT (Figure 1B) or a physiologically relevant reducing agent, NADPH (1 mM, Figure 1C), the generation of 4-MeOEN was clearly observed. Spiking the

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Figure 2. Michaelis-Menten analysis for COMT-catalyzed methylation of 4-OHEN. Various concentrations of 4-OHEN (0.5-10 µM) were incubated with 200 nM COMT for 3 min. Samples were analyzed as described in the Materials and Methods. Each data point represents the mean ( SE from six independent experiments.

product mixture with an authentic standard of 4-MeOEN (1 µM) and analysis using HPLC showed a single, more intense peak at the same retention time (data not shown), indicating that the product was 4-MeOEN generated by methylation of 4-OHEN catalyzed by COMT. LC-MS analysis confirmed that the product was 4-MeOEN, which had been fully characterized previously (data not shown, 23). Kinetic Analysis of COMT-Catalyzed Methylation of 4-OHEN. The methylation rate of 4-OHEN (0.5 or 10 µM) by 200 nM COMT was linear for 5 min (r2 > 0.98, data not shown). Concentration linearity was also observed with up to 320 nM of COMT during 3 min of incubation with 0.5, 8, or 10 µM 4-OHEN (data not shown). Therefore, all subsequent kinetic experiments were carried out for 3 min with 200 nM COMT. It should be noted that DTT (1 mM) was included in the incubations in order to prevent oxidation of 4-OHEN to 4-OHENo-quinone. Six independent experiments using various concentrations of 4-OHEN incubated with 200 nM COMT for 3 min were performed to determine the kinetic parameters (Km and kcat) of COMT with 4-OHEN as a substrate. The amount of 4-MeOEN was calculated from a standard curve, which was linear for up to 5 µM 4-MeOEN (data not shown). Figure 2 shows a MichaelisMenten plot for COMT-catalyzed methylation of 4-OHEN from which the Km and kcat were determined to be 2.4 µM and 6.0 min-1, respectively. Inhibition of COMT Activity by 4-OHEN. We observed that the amount of 4-MeOEN produced by COMT decreased after 4-OHEN exceeded 15 µM even in the presence of 1 mM DTT in the kinetic experiments (data not shown). Therefore, DTT facilitated the formation of 4-MeOEN by preventing autoxidation of 4-OHEN to the o-quinone. To investigate if 4-OHEN inhibits the methylation activity of COMT, COMT was prepared without DTT and incubated with 4-OHEN in the presence or absence of DTT. The methylation activity of COMT was then measured at different time points using 4-OHE2 as a substrate. The rate of COMT-catalyzed methylation of 4-OHE2 (100 µM) was linear for up to 5 min using 200 nM COMT and linear in COMT concentration (up to 320 nM) after a 3 min incubation (data not shown), which was consistent with previous findings (29). On the basis of these data, all inhibition experiments on the methylation of 4-OHE2 were carried out using 3 min

Figure 3. Inhibition of COMT activity by 4-OHEN. COMT (20 µM) was incubated with DMSO or various concentrations of 4-OHEN for 10 min in the presence (open circles) or absence (open squares) of 1 mM DTT or with 4-OHEN for 10 min in the absence of DTT followed by addition of 1 mM DTT for 5 min (closed triangles). The activity of COMT was measured using 4-OHE2 as the substrate as described in the Materials and Methods. Each data point represents the mean ( SE from two independent experiments.

incubations with 200 nM COMT treated with 4-OHEN or DMSO (control). Figure 3 showed that 4-OHEN inhibited the COMTcatalyzed methylation of 4-OHE2 in the absence of DTT by up to 85%. DTT (1 mM) present during the preincubation significantly prevented the 4-OHEN-mediated inhibition of COMT activity since only 30% inhibition was observed at the maximum concentration of 4-OHEN (15 µM). However, when DTT was added after a 10 min preincubation, the inhibition of COMT-catalyzed 4-OHE2 methylation was not significantly different from experiments without DTT suggesting that the inhibition was irreversible (Figure 3). To confirm this observation, gel filtration experiments were performed after treating COMT with 4-OHEN (10 µM). The 4-OHEN-mediated inhibition of COMT-catalyzed methylation activity of 4-OHE2 was then determined and found to be similar (34% of control) to experiments without gel filtration (25% of control). A kinetic study of the 4-OHEN-mediated irreversible inhibition of COMT-catalyzed methylation of 4-OHE2 showed dose- and time-dependent inhibition in the absence of DTT (data not shown). The dissociation constant for the reversible enzyme-inhibitor complex (Ki) was 26.0 ( 2.1 µM, and the rate constant for the conversion of the reversible enzyme-inhibitor complex to the irreversibly inhibited enzyme (k2) was (1.62 ( 0.13) × 10-2 s-1, calculated as described previously (20, 31) from data obtained from three independent experiments. 4-OHEN-Mediated Oxidation of COMT. Because the formation of disulfide bonds in proteins often results in the generation of intermediates that can be distinguished from the reduced forms by their altered migration mobility on nonreducing SDS-PAGE (32), we examined the formation of disulfide bonds in COMT induced by 4-OHEN using nonreducing SDS-PAGE. As compared to the control, 4-OHEN treatment generated a new 50 kDa band, which was probably a dimeric enzyme formed by intermolecular disulfide bond forma-

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Figure 4. Nonreducing SDS-PAGE of COMT modified by 4-OHEN. COMT (20 µM) was incubated with various concentrations of 4-OHEN in 50 mM ammonium bicarbonate buffer (pH 8.0) at 37 °C for 15 min. Samples (50 µL) were mixed with 10 µL of sample buffer without β-mercaptoethanol and then subjected to SDS-PAGE. The protein bands were visualized by Coomassie Brilliant Blue staining.

tion (Figure 4). The amount of the 50 kDa species increased as the concentration of 4-OHEN was increased. Several more bands with slower migration mobility appeared in samples treated with higher concentrations of 4-OHEN, possibly due to protein aggregation caused by reactive oxygen species (21, 33). Incubation with 10 mM DTT for an additional 15 min after 4-OHEN treatment of COMT resulted in conversion of the oxidized proteins to only the 25 kDa band (data not shown), which confirmed that 4-OHEN treatment caused intermolecular disulfide bond formation in COMT. Mass Spectrometry Analyses of 4-OHEN-Modified COMT. To examine if COMT was covalently modified by 4-OHEN-o-quinone, COMT (20 µM) was incubated with various concentrations of 4-OHEN and then analyzed using ESI-MS. The mass spectra showed only one abundant ion peak at m/z 24730 in the DMSO-treated COMT (Figure 5A). A minor peak (* in Figure 5A) with an m/z value 88 units higher, which might be phosphorylated COMT, was also observed. Incubation with 5 µM 4-OHEN generated a second major peak and a minor peak (I and II in Figure 5B) corresponding to COMT plus one 4-OHEN-o-quinone and COMT plus two 4-OHEN-oquinones, respectively, suggesting that at least two residues of COMT were covalently modified by 5 µM 4-OHEN. At least three residues of COMT (peaks I-III in Figure 5C) were modified when incubated with 25 µM 4-OHEN. More peaks were observed when COMT was incubated with 75 µM 4-OHEN, suggesting that additional residues were modified (data not shown). Similar results were also found by MALDI-TOF-MS analysis, further supporting that 4-OHEN alkylated multiple residues of COMT (data not shown). Peptide mapping experiments were performed using MALDI-TOF-MS analysis to identify the modified residues on COMT. As predicted, CNBr (which cleaves after methionine residues) cleavage of COMT generated more than four peptides (i-iv) as shown in Figure 6A. Peptide i represents amino acid residues 77-102 of COMT in which there was only one Cys residue (Cys95); peptide ii represents amino acid residues 103-137 of COMT without any Cys residues. Peptide iii represents amino acid residues 41-76 of COMT with only one Cys residue (Cys69); peptide iv represents amino acid residues 2-40 of COMT with only one Cys residue (Cys33). After cleavage of COMT treated with 5 µM 4-OHEN, a peak corresponding to the covalently modified COMT (iv* in Figure 6B) was detected with an m/z value approximately 280 higher than 4406. As shown in Figure 6C, 25 µM 4-OHEN treatment gave one more adduct peak (i*) in addition to the one (iv*) seen in the 5 µM 4-OHENtreated COMT. The relative intensity of the common

Figure 5. Deconvoluted positive ion electrospray ionization mass spectra of COMT modified by 4-OHEN. COMT (20 µM) was incubated with (A) DMSO, (B) 5 µM 4-OHEN, or (C) 25 µM 4-OHEN for 15 min. The samples were prepared and analyzed by ESI-MS as described in the Materials and Methods.

adduct peak (iv*) as compared with the original peptide (iv) was much higher for the 25 µM 4-OHEN-treated COMT than for the 5 µM 4-OHEN-treated COMT. Unfortunately, no significant adduct peaks were detected from trypsin digestion of 4-OHEN-treated COMT. Previously, we demonstrated that 4-OHEN inactivated human GST P1-1 by disulfide bond formation and covalent modification of Cys residues (21). It has also been shown that several Cys residues of human S-COMT are readily available for modification resulting in enzyme inactivation (34). The present study indicated that Cys33 is probably the residue covalently modified by 4-OHEN although at higher concentrations Cys95 could also be modified.

Discussion Estrogen exposure has been shown to be associated with the increased incidence of hormone-dependent

4-OHEN Is a Substrate and Inhibitor of COMT

Figure 6. Positive ion MALDI-TOF mass spectra of CNBrcleaved peptides of COMT that had been modified by 4-OHEN. COMT (20 µM) was incubated with (A) DMSO, (B) 5 µM 4-OHEN, or (C) 25 µM 4-OHEN and treated with CNBr, and the digested peptides were analyzed by MALDI-TOF as described in the Materials and Methods.

cancers in humans (35). One mechanism of estrogen carcinogenesis involves metabolism of endogenous estrogens to catechol estrogens and then oxidation to oquinones causing damage to cellular macromolecules through oxidation and alkylation resulting in toxicity. Besides glutathione conjugation, methylation by COMT is a major pathway of catechol estrogen detoxification in cells (36). Recombinant human S-COMT has been shown to methylate many endogenous and exogenous catechols with Km values that range from 0.02 to 1800 µM for the 46 catechols tested (37). COMT seems to methylate the endogenous catechol estrogens 4-OHE1 and 4-OHE2 only at the 4-OH group (29). In the present study, we found that COMT also methylated the equine catechol estrogen 4-OHEN at the 4-OH group with a Km of 2.4 µM, which was significantly lower than those reported for 4-OHE1 (53 µM) and 4-OHE2 (12-13 µM) (29, 37). Consistent with previous reports that 4-OHEN inactivated human detoxification enzymes in vitro and in cultured human cells (20-22), the present study showed that 4-OHEN also inhibited the methylation activity of human COMT in vitro. 4-OHEN inhibited the COMTcatalyzed methylation of 4-OHE2 in the absence of the reducing agent, DTT (Figure 3). The presence of DTT in the preincubation mixture restored enzyme activity of COMT (Figure 3). DTT functions as a reducing agent, which prevents autoxidation of 4-OHEN to the o-quinone protecting COMT from 4-OHEN-mediated damage. Our data (Figure 1) suggest that in a normal reducing

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environment in vivo, 4-OHEN will be primarily converted to 4-MeOEN by COMT. However, once GSH is depleted and the cellular redox status is disturbed, 4-OHEN will autoxidize to the o-quinone and it is quite likely that other toxic pathways as shown in Scheme 1 will dominate. Inhibition kinetics was studied according to Kitz and Wilson (31), since COMT activity could not be restored after gel filtration and inhibition of COMT was not reversible by 100-fold dilution. Reducing agents were not included in the preincubation mixture containing COMT and 4-OHEN. The samples were then diluted 100-fold, and COMT-catalyzed methylation of 4-OHE2 was measured. Because the dilution step removes all influence of 4-OHEN, the presence of DTT in the assay buffer only stabilizes COMT. Kinetic experiments showed that 4-OHEN inhibited methylation of 4-OHE2 in a dose-dependent manner and the inhibition progressed exponentially with time (data not shown). The Ki value of COMT with 4-OHEN was 26.0 µM, which was considerably lower than Ki values determined with other cellular enzymes with the exception of GST P1-1 (20.8 µM) (22). However, the rate of inhibition is governed by k2/Ki, which was calculated to be 622 M-1 s-1 for COMT and 67 M-1 s-1 for GST P1-1 (22), which shows that COMT is the most sensitive enzyme to 4-OHEN-mediated inhibition studied to date. The enhanced sensitivity could be due to the fact that unlike all other enzymes tested, 4-OHEN is a substrate for COMT. Of the seven Cys residues (Cys33, Cys69, Cys95, Cys157, Cys173, Cys188, and Cys191) in human SCOMT, four are present at positions identical to the rat sequence (Cys33, Cys69, Cys157, and Cys191) (38). Although no crystal structure of human COMT is available, analysis of the crystal structure of rat S-COMT indicates that Cys69, Cys95, and Cys173 may be located in the active site of human COMT including both the coenzyme binding motif and the catalytic site (38, 39). It is hypothesized that all Cys residues in human S-COMT exist as free thiols (34) and that the presence of intact thiol groups is critical for enzyme activity (40). Inactivation of human S-COMT by thiol modifying compounds may be due to the concomitant modification of several Cys residues instead of a single specific residue (34). Our study showed that one of mechanisms of 4-OHENmediated COMT inactivation involves modification of Cys residues (Scheme 1), since 4-OHEN not only induced the formation of intermolecular disulfide bonds (Figures 3 and 4) from oxidation of SH groups by free radicals but also formed covalently modified protein species (Figures 5 and 6) from alkylation of SH groups of Cys residues by 4-OHEN-o-quinone. At least two residues of COMT were modified by 4-OHEN at the 5 µM dose, and additional residues were modified as the concentration of 4-OHEN was increased (Figure 5). Previously, it was demonstrated that 4-OHEN inactivated human GST P1-1 by disulfide bond formation and covalent modification of Cys residues (21). It has also been shown that several Cys residues of human S-COMT are readily available for modification resulting in enzyme inactivation (34). Peptide mapping experiments (Figure 6) showed that Cys33 was the residue most likely covalently modified by 4-OHEN in vitro. Alkylation or/and oxidation of Cys33, which is not located in the active site of COMT (38), could lead to a confirmation change in the tertiary structure, which results in loss of enzyme activity. Although at least two

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residues of COMT were modified by 4-OHEN at the 5 µM dose and at least three residues by 4-OHEN at the 25 µM dose (Figure 5), only one and two adducts were detected after CNBr cleavage of COMT treated by 5 and 25 µM 4-OHEN, respectively (Figure 6). We have observed that GSH conjugates of 4-OHEN-o-quinone are unstable especially under basic conditions (20), and it is quite possible that certain adducts formed between the 4-OHEN-o-quinone and the Cys residues of COMT are also unstable under our experimental conditions. This hypothesis is supported by the fact that no adducts were detected after trypsin digestion of 4-OHEN-treated COMT since the trypsin is only active under basic conditions. These data do not exclude the formation of adducts with other Cys residues and/or other nucleophilic amino acid residues, which were not identified in the present study. Future X-ray diffraction and more extensive peptide mapping studies will give better insight into the structural changes of COMT after modification by 4-OHEN. In human breast cancer cells (MCF-7 and MDA-MB231), equilenin was metabolized to 4-OHEN and further methylated producing 4-MeOEN (23). Methylation of the endogenous catechol estrogen 2-OHE2 by COMT was found to protect against oxidative DNA damage caused by 2-OHE2 (41). Catechol metabolites of PCBs also inhibited the COMT-catalyzed methylation of 4-OHE2 and 2-OHE2 in vitro and in vivo (42) although the mechanism of inhibition was not investigated. Accordingly, inhibition of COMT methylation by 4-OHEN might reduce the detoxification of endogenous catechol estrogens in cells. In conclusion, 4-OHEN was not only a substrate of recombinant human soluble COMT in vitro, but it also inhibited its own methylation by COMT even in the presence of the reducing agent DTT especially at higher concentrations. 4-OHEN was an irreversible inhibitor of COMT-catalyzed methylation of 4-OHE2, and DTT significantly prevented the 4-OHEN-mediated inhibition of COMT activity. 4-OHEN not only caused the formation of intermolecular disulfide bonds but also covalently bound to multiple residues of the enzyme. Cys33 was the residue most likely modified by 4-OHEN in vitro; however, at higher concentrations, it appears that 4-OHEN also modifies Cys95. The mechanism of enzyme inhibition involved oxidation and covalent modification. These data suggest that inhibition of COMT methylation activity by 4-OHEN may reduce endogenous catechol estrogen clearance in cells thus prolonging their ability to cause oxidative damage.

Acknowledgment. This work was supported by NIH Grants CA73638 to J.L.B., CA77550 to J.D.Y., and CA83124 to R.B.v.B. We thank Dr. Wenkui Li for excellent technical assistance with LC-MS and Dr. Fagen Zhang and Linning Yu for their helpful advice on HPLC.

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