Equine Catechol Estrogen 4-Hydroxyequilenin Is a More Potent

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Equine Catechol Estrogen 4-Hydroxyequilenin Is a More Potent Inhibitor of the Variant Form of Catechol-O-Methyltransferase Yan Li,† Jiaqin Yao,† Minsun Chang,† Dejan Nikolic,† Linning Yu,† James D. Yager,‡ Andrew D. Mesecar,§ Richard B. van Breemen,† 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 Bloomberg School of 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 South Ashland Avenue, Chicago, Illinois 60607 Received November 26, 2003

Catechol-O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. It has been shown that COMT is genetically polymorphic with a wild-type and variant form where a valine has been substituted with a methionine. Several, but not all, epidemiological studies have shown that women, homozygous with the variant form, have an increased risk of developing breast cancer. Previously, we showed that 4-hydroxyequilenin (4-OHEN), a cytotoxic/genotoxic equine catechol estrogen metabolite, is both a substrate of COMT and an irreversible inhibitor of the methylation activity of COMT in vitro. To further understand the mechanism(s) of the association between the breast cancer risk and the COMT polymorphism, it was of interest to study the effect of the Val/Met polymorphism on COMT-catalyzed catechol estrogen methylation and 4-OHEN-mediated inhibition. In the present study, Michaelis-Menten analysis showed no difference between the relative ability of each form to methylate 4-OHEN. However, we found that the COMT variant form was more susceptible to 4-OHEN-mediated irreversible inactivation. Electrospray ionization mass spectrometry and SDS-gel analysis of COMT modified by 4-OHEN revealed that inhibition mechanisms include alkylation and/or oxidation of certain amino acids. In addition, site-directed mutagenesis experiments showed that Cys33 played a more important role in the variant form of COMT demonstrated by the fact that the C33A mutant of the variant form of COMT decreased its catalytic capability more dramatically as compared with that of wild type. Furthermore, thermotropic studies indicated that the variant form was more thermolabile, which suggested that the valine to methionine substitution may have changed the secondary/tertiary structure of the variant form of COMT, making it more susceptible to 4-OHEN and heat inactivation. These data suggest that 4-OHEN-mediated inhibition of the variant form of COMT in vivo might affect the detoxification efficiency of endogenous and/or exogenous catechol estrogens and play a role in the association between breast cancer risk and COMT polymorphism.

Introduction COMT1 (EC 2.1.1.6) catalyzes the transfer of a methyl group from the donor SAM to a catechol substrate (1). In mammals, COMT is widely distributed in brain and * 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 Bloomberg School of Public Health. § Center for Pharmaceutical Biotechnology (M/C 870), University of Illinois at Chicago. 1 Abbreviations: DTT, dithiothreitol; ESI-MS, electrospray ionization mass spectrometry; GST, glutathione S-transferase; MB-COMT, membrane-bound catechol-O-methyltransferase; PBS, phosphatebuffered saline; PCR, polymerase chain reaction; 2-MeOE2, 2-methoxyestradiol; 4-MeOE2, 4-methoxyestradiol; 4-MeOEN, 4-methoxyequilenin; 2-OHE2, 2-hydroxyestradiol; 4-OHE2, 4-hydroxyestradiol; 4-OHEN, 4-hydroxyequilenin; SAM, S-adenosyl-L-methionine; SCOMT, soluble catechol-O-methyltransferase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

peripheral tissues (2). The physiological substrates of COMT include a wide variety of chemical compounds such as catecholamine neurotransmitters and endogenous and exogenous catechol estrogens (3, 4). It has been a long-held view that the major physiological function of the COMT is primarily for the inactivation of biologically active and toxic endogenous and/or exogenous catechols (1). There are two forms of COMT, S-COMT and MB-COMT, which are encoded by a single gene with different transcription start sites. S-COMT is the predominantly expressed form in most human tissues (5). COMT activity varies among individuals (6), and the level of COMT enzyme activity is genetically polymorphic with a trimodal distribution of low, intermediate, and high levels of activity in red blood cells and liver (7). This genetic polymorphism results in 3-4-fold differences in COMT activity (7). More recently, it was found that both

10.1021/tx0342464 CCC: $27.50 © 2004 American Chemical Society Published on Web 03/06/2004

Selective Inhibition of Variant COMT by 4-OHEN

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

a

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

S-COMT and MB-COMT have a wild-type (Val108 in S-COMT and Val158 in MB-COMT) and at least one variant form (Met108 in S-COMT and Met158 in MB-COMT) and that the valine to methionine substitution was responsible for the varied COMT activity in erythrocytes (8). Therefore, it has been hypothesized that the variant form of COMT may have a lower capacity than the wild type to methylate catechol estrogens, and thus, the wild-type and variant forms of COMT were designated as “high activity” and “low activity” forms, respectively (8). In support of this, several, but not all, epidemiological studies have shown that women, homozygous with the variant form of COMT, have an increased risk of developing breast cancer (9-13). There is a clear association between excessive exposure to synthetic and endogenous estrogens and the development of cancer in several tissues (14-16). 4-OHEN, which is a major phase I metabolite of several equine estrogens present in the most popular estrogen replacement formulation, is considerably cytotoxic/genotoxic. 4-OHEN rapidly autoxidizes to electrophilic/redox active o-quinone, which can damage cells through a variety of different pathways (Scheme 1). Previously, we found that 4-OHEN is a potent irreversible inactivator of GST (17) and COMT (18) by covalently modifying SH groups on cysteine residues. In addition, 4-OHEN-o-quinones undergo redox cycling with their semiquinone radicals to generate excessive reactive oxygen species (19, 20), which induce oxidative cleavage of the DNA phosphate-sugar backbone, oxidation of the purine/pyrimidine residues of DNA, and DNA single strand breaks (17, 19-24). Because of the association of COMT with breast cancer risk, it is of interest to characterize the effects of the Val/ Met polymorphism on the methylation of catechol estrogens in vitro. Previously, we have shown that 4-OHEN is not only a substrate of human recombinant COMT but also an irreversible inhibitor of COMT-catalyzed methylation of endogenous and exogenous catechol estrogens

(18). To further investigate whether the methylation rates of 4-OHEN by the wild-type and variant forms of COMT are different, kinetic studies were performed with these two isoforms. Michaelis-Menten kinetic analysis showed no difference between the relative ability of each form to methylate 4-OHEN. However, we found that the variant form of COMT was more susceptible to 4-OHENmediated irreversible inactivation as compared to the wild type. Site-directed mutagenesis data showed that Cys33 may play a more important role in variant form of COMT. Furthermore, thermotropic studies indicated that the variant form of COMT was more thermolabile, which suggests that the Val to Met substitution may change the secondary/tertiary structure of the variant form of COMT making it more susceptible to inhibition. Our data may provide a partial explanation for the association between breast cancer risk and COMT polymorphism.

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 (20). 4-MeOEN was synthesized as described previously (28, 29) and used as a standard for the quantitative analysis of COMT methylation. Recombinant human wild-type and variant forms of S-COMT were prepared as described previously (30) and stored in PBS with 7% glycerol and 1 mM DTT at -80 °C. Enzyme Activity Assays and Kinetic Measurements. COMT activity was determined by using HPLC as described previously (30, 31) 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

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of 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 at different temperatures 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 concentrations of the other reagents were the same 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 Inc., Chicago, IL). HPLC. 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 autoinjector, SPD-M10AV UV/vis photodiode array detector (280 nm), and SPD-10AV detector (Shimadzu Scientific Instruments, 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. 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 or 45 °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. Aliquots (5 µL) were removed at various times and diluted 100-fold into the assay buffer [200 mM sodium phosphate buffer (pH 7.8), 5 mM MgCl2, 1 mM DTT, and 0.2 mM SAM] 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. Inhibition kinetic studies were performed with 2-50 µM 4-OHEN according to Kitz and Wilson (32). Irreversible kinetic parameters were obtained using the following equations where it is assumed that [I] . [Eo]. [I] is the inhibitor concentration, [Eo] is the concentration of COMT at time 0, EI is the enzyme inhibitor complex, and E* is the inactivated enzyme. Ki and k2 were obtained from double-reciprocal plots (i.e., eq 2) of the apparent first-order rate constant vs inhibitor concentration. Ki

k2

I + E y\z EI 98 E* kapp )

k2 1+

Ki

(1)

[I]

Ki 1 1 ) + kapp k2 k2[I]

(2)

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 various times. In some cases, 10 mM DTT was further incubated with 4-OHEN-treated COMT for an additional 15 min to determine if 4-OHEN-induced disulfide bond formation could be irreversible. Treated samples (50 µL) were mixed with 10 µL of sample buffer without any reducing agent, followed by incubation at 100 °C for 5 min, and then subjected to SDSPAGE. 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 and analyzed by infusion at 20 µL/min using positive ion ESI-MS on a Quattro II triple quadrupole mass spectrometer (Waters, Boston, MA). In Vitro Site-Directed Mutagenesis. Site-directed mutagenesis for the Cys33 position of both wild-type and variant forms of COMT was carried out as recommended by the manufacturer (Stratagene, La Jolla, CA). Briefly, QuikChange Site-Directed Mutagenesis Kit was used and overlapping PCR was carried out using pairs of mutually complementary primer containing mismatching bases (underlined) for the Cys33 codon (sense strand, 5′-GGCCATTGACACCTACGCCGAGCAGAAGGAGTGGG-3′; antisense strand, 5′-CCCACTCCTTCTGCTCGGCGTAGGTGTCAATGGCC-3′). The oligonucleotide primers, each complementary to opposite strands of the COMT gene sequence, were extended during temperature cycling (the reactions were first incubated at 95 °C for 30 s, and the PCR itself consisted of 16 cycles with 30 s at 95 °C, 1 min at 55 °C, and 10 min at 68 °C) by using PfuTurbo DNA polymerase. Incorporation of the oligonucleotide primers generates the mutants with cysteine to alanine substitution (C33A) for both wild-type and variant forms of COMT. Gene sequences of the mutated wildtype and variant form of COMT were confirmed by DNA sequence analysis by the Research Resources Center DNA Sequencing Facility at the University of Illinois at Chicago. Recombinant human mutated wild-type and variant forms of S-COMT were prepared as described previously (30). Kinetic studies of wild-type and mutant COMT enzymes were conducted using both endogenous (4-OHE2) and exogenous (4-OHEN) catechols as substrates. Thermotropic Properties of COMT. The thermotropic properties of the recombinant wild-type and variant forms of COMT were characterized at 37 and 45 °C. The wild-type and variant forms of COMT were incubated at 37 or 45 °C in 200 mM potassium phosphate buffer (pH 7.8) in the presence or absence of 10 mM DTT. 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, and the remaining samples were subjected to electrophoretic analyses as described above.

Results Kinetic Analysis of COMT-Catalyzed Methylation of 4-OHEN. Initial studies were performed to optimize the assay conditions for pH, sodium phosphate buffer, MgCl2 concentration (30), COMT concentration, and incubation time (18). According to the results, all 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-OHEN-o-quinone. Previously, we found that human recombinant wild type of COMT can methylate 4-OHEN at the 4-OH group with a low Km value as compared to other endogenous catechol estrogens (18). To further investigate whether the wild-type and variant forms of COMT have the same capability to methylate 4-OHEN, five independent experiments using various concentrations of 4-OHEN incubated with 200 nM wildtype or variant form were performed. The amount of 4-MeOEN was calculated from a standard curve, which was linear for up to 16 µM 4-MeOEN (data not shown). Figure 1 shows a Michaelis-Menten plot for the wildtype and variant forms of COMT-catalyzed methylation of 4-OHEN at 37 °C. No significant differences were observed between the kinetic parameters of the wild-type and variant forms of COMT (Figure 1 and Table 1). Furthermore, the kinetic properties of recombinant COMT were also characterized at 20 and 45 °C. Again, no



Figure 1. Michaelis-Menten analysis for the wild-type (closed squares) and variant forms (closed triangles) of COMT-catalyzed methylation of 4-OHEN at 37 °C. Various concentrations of 4-OHEN (0.5-50 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 ( SD of five independent experiments. Table 1. Kinetic Constants for O-Methylation of 4-OHEN by Wild-Type and Variant Forms of COMT at Different Temperatures 20 °C kcat (min-1)

wild type variant form Km (µM) wild type variant form kcat/Km (min-1 µM-1) wild type variant form

37 °C

45 °C

7.9 ( 0.2a 25.3 ( 1.8 68.0 ( 19.6 8.1 ( 0.2 22.9 ( 1.7 90.3 ( 14.1 3.4 ( 0.3 9.9 ( 1.6 34.3 ( 15.0 3.4 ( 0.3 9.5 ( 1.6 46.7 ( 10.2 2.3 2.5 2.0 2.3 2.4 1.9

a Mean ( standard deviation calculated from five independent experiments.

significant kinetic differences were observed between these two COMT forms (Table 1). Inhibition of COMT Activity by 4-OHEN. Previously, we found that 4-OHEN inhibited the methylation activity of wild type COMT in vitro. Specifically, 4-OHEN inhibited the wild type of COMT-catalyzed methylation of endogenous catechol estrogen 4-OHE2 in the absence of the reducing agent, DTT. Kinetic experiments also showed that 4-OHEN inhibited methylation of 4-OHE2 in a dose-dependent manner and the inhibition progressed exponentially with time (18). To further investigate the inhibitory effect of 4-OHEN on the variant form of COMT, irreversible inhibition kinetic experiments were performed with the variant form of COMT. The kinetic study of the 4-OHEN-mediated irreversible inhibition of COMT-catalyzed methylation of 4-OHE2 showed a dose- and time-dependent inhibition in the absence of DTT for both of the wild type and variant forms of COMT (Figure 2). Table 2 shows the dissociation constant for the reversible enzyme-inhibitor complex (Ki) and the rate constant for the conversion of the reversible enzymeinhibitor complex to the irreversibly inhibited enzyme (k2) of wild-type and variant forms of COMT, calculated from data obtained in three independent experiments as described previously (17, 32). Our in vitro inhibition kinetics parameters showed that the variant form had a 2-fold lower dissociation constant Ki value as compared with the wild type, indicating that the variant form is more sensitive to 4-OHEN-induced irreversible inactivation. To investigate if heat plays a role on the 4-OHENmediated COMT inhibition, similar inhibition kinetic

Figure 2. Inhibition of recombinant (A) wild-type and (B) variant forms of COMT by 4-OHEN in vitro. A 20 µM concentration of COMT was incubated with 8 (closed squares), 15 (open circles), 25 (open triangles), 35 (open diamonds), and 45 µM (open squares) 4-OHEN for various times in the absence of DTT. Aliquots were removed and diluted 100-fold into assay buffer. The methylation activity of COMT was measured using 4-OHE2 as substrate as described in the Materials and Methods. Data represent the mean of four independent experiments. Table 2. Inhibition Kinetic Constants of Wild-Type and Variant Forms of COMT at Different Temperatures k2 (× 103 s-1) Ki (µM) k2/Ki (s-1 M -1)

wild type variant form wild type variant form wild type variant form

37 °C

45 °C

9.3 ( 1.3a 10.3 ( 0.9 13.2 ( 1.9 6.4 ( 0.8 702.5 1613.1

20.9 ( 1.9 20.3 ( 2.8 14.5 ( 1.3 4.1 ( 0.6 1447.7 4973.1

a Mean ( standard deviation calculated from four independent experiments.

experiments were conducted at 45 °C (Table 2). More than a 3-fold difference of the dissociation constants between the wild-type and the variant form was observed, which was much more significant as compared with those values observed at 37 °C. Mass Spectrometric Analyses of 4-OHEN-Modified COMT. Previously, our data showed that one of the mechanisms of 4-OHEN-mediated COMT inactivation involves modification of cysteine residues. At least two

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Figure 3. Positive ion electrospray ionization mass spectra of wild-type (left column) and variant forms (right column) of COMT modified by 4-OHEN. COMT (20 µM) was incubated with (A) DMSO, (B) 10 µM 4-OHEN, or (C) 60 µM 4-OHEN for 15 min. The samples were prepared and analyzed using ESI-MS as described in the Material and Methods. I, II, III, and IV indicate the number of 4-OHEN-o-quinone molecules that covalently modified proteins.

cysteine residues of COMT were modified by 4-OHEN at low concentration, and more cysteine residues were modified as the concentration of 4-OHEN was increased (18). Because the kinetics of the irreversible inhibition showed that the variant form of COMT was more sensitive to 4-OHEN-mediated inhibition, our initial expectation was that more alkylation should be observed in the variant form of COMT samples. Contrary to our expectations, ESI-MS analysis showed similar alkylation patterns of the wild-type and variant forms of COMT by 4-OHEN-o-quinone (Figure 3). Kinetic Properties of Wild-Type and Mutant COMT. Our previous data showed that Cys33 is the first amino acid alkylated by 4-OHEN-o-quinone (18); to investigate the role of Cys33 played in the catalytic process, C33A mutants of both wild-type and variant forms of COMT were obtained using an in vitro sitedirected mutagenesis technique. Kinetic studies of wildtype and mutant enzymes using both endogenous and

exogenous catechols 4-OHE2 and 4-OHEN were conducted at 37 °C (Figure 4). Kinetic data showed that substitution of Cys33 with alanine in both wild-type and variant forms of COMT decreased their catalytic capabilities (Table 3) demonstrated by lower kcat values although the substitution did not affect their affinities (Km) toward substrates. Interestingly, the mutated variant form of COMT decreased its catalytic capability much more dramatically as compared with the mutated wild type of COMT for both endogenous (4-OHE2) and exogenous (4-OHEN) (50% decrease for wild-type and 75% decrease for variant form of COMT) catechol substrates. 4-OHEN-Mediated Oxidation of COMT. The formation of inter- and/or intramolecular disulfide bonds from oxidation of SH groups by free radicals might also be one of the mechanisms of enzyme inactivation. 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 mobility on nonreducing



Figure 5. Nonreducing SDS-PAGE of (A) wild-type and (B) variant forms of COMT modified by 4-OHEN. COMT (20 µM) was incubated with 32 µM 4-OHEN in 50 mM ammonium bicarbonate buffer (pH 8.0) at 37 °C for various times. 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. Lane 1, wild-type control; lane 2, variant form control; lane 3, wild-type incubated for 1 min; lane 4, variant form incubated for 1 min; lane 5, wild-type incubated for 2 min; lane 6, variant form incubated for 2 min; lane 7, wild-type incubated for 3 min; and lane 8, variant form incubated for 3 min.

Figure 4. Kinetic analysis for the wild-type (closed squares) and variant forms (closed circles) of COMT and their C33A mutants (open squares and circles)-catalyzed methylation of (A) 4-OHEN and (B) 4-OHE2 at 37 °C. Various concentrations of 4-OHEN (0.5-25 µM) and 4-OHE2 (10-150 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 ( SD of three independent experiments. Table 3. Kinetic Constants for O-Methylation of 4-OHEN by Wild Types and C33A Mutants COMT at 37 °C kcat (min-1) wild type 22.6 ( 1.9a variant form 21.4 ( 1.4 C33A mutant of wild type 11.0 ( 0.3 C33A mutant of variant form 5.9 ( 0.4

Km (µM)

kcat/Km (min-1 µM -1)

8.5 ( 1.6 8.0 ( 1.2 9.9 ( 0.7 8.7 ( 1.4

2.7 2.7 1.6 0.7

a Mean ( standard deviation calculated from three independent experiments.

SDS-PAGE (33). Previously, we observed that wild-type COMT treated with 4-OHEN generated a new 50 kDa band, which was a dimeric enzyme formed by intermolecular disulfide bond formation (18). In the present study, the variant form of COMT treated with 4-OHEN also generated this 50 kDa band. Interestingly, the variant form generated more enhanced 50 kDa bands than the wild type under the same experimental conditions after 1, 2, and 3 min of incubation with 4-OHEN (Figure 5), which suggests that there is more enzyme damage resulting from the oxidation of the variant form after 4-OHEN treatment. This might partially account

Figure 6. Thermotropic properties of wild-type (closed squares) and variant forms (closed triangles) of COMT. COMT (20 µM) samples were incubated at 45 °C in the absence of any reducing agent while control samples were kept on ice. The heated samples were placed on ice for 5 min prior to measurement of enzyme activity as described in the Material and Methods. All measured activities were normalized as percentage of control activity. Each data point represents the mean ( SD of two independent experiments.

for the different sensitivity to 4-OHEN-mediated inactivation between the wild type and the variant form of COMT. However, no significant difference was observed for longer incubation time between the wild type and the variant forms of COMT. Thermotropic Properties of Recombinant COMT. It has been shown that the variant form of COMT is more thermolabile than the wild type (31, 34). Because of these observations, we compared the thermolability of these two COMT forms. As shown in Figure 6, 4-OHE2 methylation by the variant form of COMT was reduced more than the wild type during a 20 min incubation at 45 °C. This trend was more obvious for longer incubation times of 40 and 60 min (Figure 6). When these heated COMT samples were subjected to nonreducing SDS-PAGE, both of the two COMT forms generated 50 kDa bands. Interestingly, the intensity of the 50 kDa bands generated by the heated variant form of COMT was more enhanced than those formed by the heated wild type (data not shown).

Discussion Known risk factors for women developing breast cancer are associated with estrogen exposure including long-

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term estrogen replacement therapy. 4-OHEN is the major phase I catechol metabolite of the equine estrogens equilenin and equilin, which can constitute about 50% of the most widely prescribed estrogen replacement formulation, Premarin (Wyeth-Ayerst) (19, 35). One mechanism of estrogen carcinogenesis involves metabolism of endogenous and exogenous estrogens to catechols and then oxidation to o-quinones causing damage to cellular macromolecules through oxidation and alkylation. There are at least two physiological detoxification pathways for o-quinones or catechols in human cells. First, physiological nucleophiles in cells such as glutathione can readily form conjugates with o-quinones. Another major detoxification pathway of the carcinogenic catechol estrogens is O-methylation catalyzed by COMT. It has been shown that COMT activity varies widely among individuals (6) and Lachman et al. showed that polymorphism of COMT was responsible for different COMT activity (8). The association of COMT polymorphism and breast cancer risk is still controversial. Several epidemiological studies have shown that women homozygous with the variant COMT form have an increased risk of developing breast cancer (9-12). In particular, Mitrunen et al. showed that increased breast cancer risk was observed for postmenopausal women with the homozygous variant form of COMT genotype and long-term (>30 months) use of estrogen (OR 4.02) (13). However, some other studies have shown that COMT polymorphism is not associated with increased risk of developing cancers (36, 37). In light of these considerations, our initial hypothesis was that the variant form of COMT had a lower capability to methylate toxic catechol estrogens, which could increase breast cancer risk. However, in our present studies, we found that no significant differences were observed between the kinetic parameters of recombinant wild-type and variant forms of COMT. Similarly, Goodman et al. also showed that the kinetic parameters were similar for recombinant wild-type and variant forms of COMT using endogenous catechol estrogens as substrates (30). Furthermore, kinetic studies conducted at different temperatures showed no significant kinetic differences between these two COMT forms. These data suggest that the catalytic efficiency of the Met variant form was similar to that of the Val wild type with respect to methylation of 4-OHEN. Therefore, the association of COMT polymorphism with breast cancer risk is probably not due to the different detoxification efficiencies. Previously, we showed that 4-OHEN is both a substrate of COMT and an irreversible inhibitor of the methylation activity of COMT in vitro. 4-OHEN inhibited the COMT-catalyzed methylation of 4-OHE2 in the absence of reducing agents (18). To determine if 4-OHEN had similar inhibitory effects on the wild-type and variant forms of COMT, inhibition kinetics were evaluated at 37 °C according to the method of Kitz and Wilson (32). For both wild-type and variant forms of COMT, the kinetic study of the 4-OHEN-mediated inhibition of COMT-catalyzed methylation activity of 4-OHE2 showed dose- and time-dependent inhibition in the absence of any reducing agent. Although there was no significant difference between the rate constants for the conversion of the reversible enzyme-inhibitor complex to the irreversibly inhibited enzyme (k2) between the wild-type and variant forms of COMT, we did find that the variant form of COMT had a 2-fold lower Ki value as compared to the wild type (Table 2), suggesting that the variant form had

Li et al.

a higher affinity for 4-OHEN-o-quinone and thus higher sensitivity to 4-OHEN-mediated irreversible inhibition. Furthermore, the rate of inhibition is governed by k2/Ki, which was calculated to be 1613 M-1 s-1 for the variant form, almost 2-fold higher than 702 M-1 s-1 for the wild type. Similar inhibition experiments conducted at 45 °C showed more than 3-fold difference in the k2/Ki value between wild-type and variant forms of COMT. Previously, we found that the wild type of COMT is the most sensitive enzyme to 4-OHEN-mediated inhibition studied to date (18). The data from the present study showed that the variant form of COMT is much more sensitive to 4-OHEN-mediated inhibition as compared to the wild type. In a normal physiological environment in vivo, reducing agents can prevent autoxidation of 4-OHEN to the o-quinone, protecting COMT from 4-OHENo-quinone-mediated damage, and 4-OHEN will be converted primarily to 4-MeOEN by either the wild-type or the variant form of COMT. However, once the normal cellular redox status is disturbed, 4-OHEN could easily be autoxidized to the o-quinone so that toxic pathways resulting from o-quinone might dominate. According to our present study, under such conditions, the variant form is much more susceptible to 4-OHEN-o-quinonemediated inhibition resulting in decreased detoxification efficiency of catechols catalyzed by the variant form of COMT. The decreased clearance of the toxic catechols will accumulate leading to more cell damage, which might account for the increased risk for breast cancer among women homozygous with variant form of COMT. We have previously shown that 4-OHEN can cause damage to COMT through an oxidative and/or alkylation mechanism(s). Alkylation of cysteine residues plays an important role in enzyme inactivation. Oxidation of COMT also leads to formation of disulfide bonds resulting in loss of enzyme activity (18). Because the variant form of COMT was more susceptible to 4-OHEN-mediated inhibition, it was of interest to examine possible mechanisms for this different sensitivity. COMT samples preincubated with 4-OHEN in the absence of reducing agents were analyzed using ESI-MS. Contrary to our expectations, these two COMT forms showed similar alkylation patterns after 4-OHEN treatment. Because our previous data (18) showed that of the seven cysteine residues (Cys33, Cys69, Cys95, Cys157, Cys173, Cys188, and Cys191) in human S-COMT, Cys33 is the most susceptible sulfhydryl group to covalent modification in both wild-type and variant forms of COMT in vitro, Cys33 is the likely target of chemical modification leading to rapid enzyme inactivation. To study whether cysteine residues in COMT are involved in the mechanism of enzyme inactivation mediated by 4-OHEN, we prepared C33A mutant proteins. Kinetic studies using both endogenous and exogenous catechol substrates showed that substitution of Cys33 with alanine in both wild-type and variant forms of COMT changed their catalytic capabilities as demonstrated by lower kcat values. More importantly, the mutated variant form of COMT showed much more dramatically decreased catalytic capability (75% decrease of kcat/Km value) as compared with mutated wild-type COMT (50% decrease of kcat/Km value). These data suggest that Cys33 may play a more important role in the variant form of COMT in that substitution with alanine or alkylation by 4-OHEN-o-quinone in this enzyme leads to more dramatically decreased catalysis. Furthermore, the different


susceptibility to Cys33 substitution/modification in the wild-type and variant forms of COMT indicates that these two enzymes might have different secondary/ tertiary structures in the local area surrounding this residue. There are no crystal structures of human COMT to confirm our hypothesis to date. Future X-ray diffraction studies will give better insight into the different structural changes of human soluble wild-type and variant forms of COMT after modification by 4-OHEN. Interestingly, when the 4-OHEN-treated enzyme samples were subjected to nonreducing SDS-PAGE, the variant form of COMT samples generated a more intense 50 kDa band than the wild type, which suggests enhanced disulfide bond formation for the variant form after 4-OHEN treatment. On the basis of these data, the enhanced sensitivity of the variant form of COMT to 4-OHEN could be due to the fact that it was more susceptible to 4-OHEN-mediated oxidative damage. It should be noted that oxidative damage to proteins is not compound specific. Under physiological conditions, hydroxyl radical, peroxy radical, and hydrogen peroxide are formed as a result of normal oxidative metabolism. It is possible that the variant form of COMT is more easily damaged even under normal physiological conditions. There is only one encoding base difference between the wild type and the variant forms of COMT. Because this single nucleotide polymorphism makes the variant form more susceptible to 4-OHEN-mediated inhibition, there may be secondary/tertiary structural change resulting from Val to Met substitution. Previous reports have shown that the variant form of COMT is more thermolabile than the wild type (8). In the present study, we found that the variant form of COMT lost its activity more rapidly as compared to the wild type after heat treatment; the loss of activity might have resulted from dimer formation as demonstrated by nonreducing SDSPAGE. These data suggest that these two COMT forms might have differences in their secondary/tertiary structures resulting in different thermolabilities. Because of the Val to Met substitution, the variant form of COMT probably has a less stable secondary and/or tertiary structure, making it more susceptible to 4-OHEN-mediated disulfide bond formation as well as changes in the secondary and/or tertiary structure leading to more enhanced enzyme inactivation. In conclusion, contrary to our initial hypothesis, the wild-type and variant forms of COMT catalyze the methylation of 4-OHEN with equal activity, which suggests that there is no functional difference between their detoxification efficiency. However, the variant form of COMT was more susceptible to 4-OHEN-mediated inhibition due to enhanced disulfide bond formation after 4-OHEN treatment as compared with the wild type. In vitro site-directed mutagenesis showed that Cys33 plays a more important role in the variant form of COMT, which suggests that the wild-type and variant forms of COMT may have different secondary/tertiary structures in the local area surrounding Cys33. Taken together, the variant form of COMT might be more susceptible to 4-OHEN-mediated inhibition resulting in reduced endogenous and exogenous catechol estrogen clearance in cells, thus prolonging their ability to cause toxicity.

Acknowledgment. This work was supported by NIH Grants CA73638 to J.L.B., CA77550 to J.D.Y., and CA83124 to R.B.vB.


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Equine Catechol Estrogen 4-Hydroxyequilenin Is a More Potent

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