Biochemical and Molecular Modeling Studies of the O-Methylation of

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) catalyzes the O-methylation of a wide array of catechol-containing substrates using s-adenosyl-l-methi...
1 downloads 0 Views 1MB Size
Download PDF
Chem. Res. Toxicol. 2007, 20, 1409–1425

1409

Biochemical and Molecular Modeling Studies of the O-Methylation of Various Endogenous and Exogenous Catechol Substrates Catalyzed by Recombinant Human Soluble and Membrane-Bound Catechol-O-Methyltransferases† Hyoung-Woo Bai,‡ Joong-Youn Shim,§ Jina Yu,‡ and Bao Ting Zhu*,‡ Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, UniVersity of Kansas Medical Center, Kansas City, Kansas 66160 and J. L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central UniVersity, Durham, North Carolina 27707 ReceiVed May 20, 2007

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) catalyzes the O-methylation of a wide array of catechol-containing substrates using S-adenosyl-L-methionine as the methyl donor. In the present study, we have cloned and expressed the human soluble and membrane-bound COMTs (S-COMT and MBCOMT, respectively) in Escherichia coli and have studied their biochemical characteristics for the O-methylation of representative classes of endogenous catechol substrates (catecholamines and catechol estrogens) as well as exogenous catechol substrates (bioflavonoids and tea catechins). Enzyme kinetic analyses showed that these two recombinant human COMTs are functionally active, with catalytic and kinetic properties nearly identical to those of crude or purified enzymes prepared from human tissues or cells. Kinetic parameters for the O-methylation of various substrates were characterized. In addition, computational modeling studies were conducted to better understand the molecular mechanisms for the different catalytic behaviors of human S- and MB-COMTs with respect to S-adenosyl-L-methionine, various substrates, and also the regioselectivity for the formation of mono-methyl ether products. Our modeling data showed that the binding energy values (∆G) calculated for most substrates agreed well with the measured kinetic parameters. Also, our modeling data precisely predicted the regioselectivity for the O-methylation of these substrates at different hydroxyl groups, the predicted values matched nearly perfectly with the experimental data. Introduction 1

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) , an enzyme ubiquitously present in mammals including humans and rodents, catalyzes the O-methylation of a wide variety of endogenous and exogenous catecholic substrates using Sadenosyl-L-methionine (AdoMet3) as the methyl donor (1–5) (depicted in Figure 1, upper panel). The human COMT exists in two different forms, namely, the soluble form (S-COMT) and the membrane-bound form (MB-COMT). In most human tissues, the majority of COMT is present as S-COMT and only a small fraction as MB-COMT. However, in human brain, approximately 70% of all COMT proteins are MB-COMT, and only about 30% of them are S-COMT (2, 3, 6). The human S-COMT protein is composed of 221 amino acids, with a molecular weight of ∼24.4 kD (6). The human MBCOMT protein contains 50 additional amino acid residues at the N-terminus, about 20 of them serving as a hydrophobic * To whom correspondence should be addressed. Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, MS-1018, room KLSIC-4061, 2146 W. 39th Ave., Kansas City, KS 66160. Phone: 913-588-9842. Fax: 913-588-7501. E-mail: BTZhu@ kumc.edu. † A major part of the research work described here was completed when H.-W.B., J.Y., and B.T.Z. worked at the University of South Carolina, Columbia, SC 29208. ‡ University of Kansas Medical Center. § North Carolina Central University. 1 Abbreviations: COMT, catechol-O-methyltransferase; AdoMet, Sadenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine; 2-OH-E2 and 4-OH-E2, 2- and 4-hydroxyestradiol, respectively.

membrane anchor (6, 7). It was found that one single gene (localized to chromosome 22, band q11.2 (8, 9)) in humans encodes both S-COMT and MB-COMT proteins by using two separate promoters (6). The gene contains six exons with the first two being noncoding, and the expression of the COMT gene is controlled by two distinct promoters located in exon 3 (6, 10, 11) (depicted in Figure 2, upper panel). The expression of the shorter 1.3-kb mRNA transcript is regulated by the P1 promoter, which is located immediately before its start codon S-ATG and overlaps the start codon for MB-COMT (MB-ATG) and also part of its coding sequence in exons 2 and 3. The synthesis of the 1.5-kb human mRNA is regulated by the distal 5′ P2 promoter. COMT catalyzes the O-methylation of various endogenous and exogenous catechols in the body (1, 2, 5, 10, 13–16). In the central nervous system, COMT metabolically deactivates catecholamine neurotransmitters (dopamine and norepinephrine) through O-methylation. Inhibition of this methylation process has been suggested to have a potentially harmful effect contributing to an increased risk of Parkinson’s disease (5, 17). In addition to catecholamine neurotransmitters, the endogenous catechol estrogens, such as 2- and 4-hydroxyestradiol (2-OHE2 and 4-OH-E2), which are major oxidative metabolites of E2 formed by cytochrome P450 isoforms in humans (18, 19), are also rapidly O-methylated by COMT, in a manner analogous to the O-methylation of catecholamines (20). COMT also catalyzes the metabolic O-methylation of various exogenous catechol-containing substrates (1, 2, 4, 5, 12, 14–16).

10.1021/tx700174w CCC: $37.00  2007 American Chemical Society Published on Web 09/20/2007

1410 Chem. Res. Toxicol., Vol. 20, No. 10, 2007

Bai et al.

Figure 1. Upper panel: COMT-mediated O-methylation of catechol substrates to form two mono-methyl ether products. Lower panels: chemical structures of four representative classes of substrates used in the present study, which include catecholamines (dopamine, epinephrine, and norepinephrine), catechol estrogens (2-OH-E2 and 4-OH-E2), tea catechins (catechin and epicatechin), and bioflavonoids (quercetin and fisetin). The two catehcolic hydroxyl groups of each substrate are labeled with empty arrowheads, and their positions were also selectively labeled with numbers for the convenience of description of the regioselective methylation reactions.

Many earlier studies have used the crude cytosolic or microsomal fractions to determine the kinetic properties of the human S-COMT and MB-COMT for the methylation of a variety of endogenous and exogenous catecholic substrates. In some of the additional studies, human S-COMT and/or MBCOMT were expressed in Escherichia coli (E. coli), insect cells, or mammalian cell lines (21–25), and the kinetic properties of the expressed COMT proteins were assessed using catecholamine neurotransmitters alone as substrates. In the present study, we have separately expressed the human S-COMT and MBCOMT in E. coli and have systematically compared the

biochemical characteristics of these two enzymes for the O-methylation of various endogenous and exogenous catechol substrates selected from four representative classes (catecholamines, catechol estrogens, tea catechins, and bioflavonoids). The chemical structures of these substrates are shown in Figure 1 (lower panels). In addition, on the basis of the available X-ray crystal structure of the rat S-COMT, detailed computational modeling studies were conducted to better understand the precise molecular mechanisms of the different catalytic behaviors of human S- and MB-COMTs with respect to AdoMet (the methyl donor), various substrates, and their regioselectivity for the

Kinetic and Molecular Studies of Human COMTs

Chem. Res. Toxicol., Vol. 20, No. 10, 2007 1411

Figure 2. Upper panel: structure of human COMT gene. The boxes represent exons, and the thin lines in between the boxes represent introns. The hatched boxes indicate the protein coding regions. The size of each exon and intron is as indicated. The positions of the initiation codons for the transcription of S-COMT and MB-COMT mRNAs are indicated as S-ATG and MB-ATG. The two known promoters, P1 and P2, are shown by black bars. Note that the P1 promoter for the transcritpion of S-COMT overlaps with the initiation codon and part of the coding sequence for MB-COMT. Lower panel: construction of the pET12a/S-COMT and pET12a/MB-COMT expression vectors based on the vector pET12a. The S-COMT or MB-COMT cDNA was cloned into the NdeI and BamH I sites of pET12a to form the pET12a/S-COMT or pET12a/MB-COMT expression vectors. Each of the expression vectors was under the control of the T7 promoter and lacO-operator. Expression was induced by the addition of 0.5 mM isopropylthio-β-D-galactoside.

formation of mono-methyl ether products. It is of note that the use of molecular modeling tools to probe the interactions of COMTs with various substrates is of unique value since each of the substrates can bind to the enzyme in two different conformations (leading to the formation of two different monomethyl ether products), and this reality makes it nearly impossible to use the current X-ray crystallographic tools to determine the precise binding structures of each substrate–enzyme complex in two coexisting conformations.

Materials and Methods Chemicals. Dopamine, epinephrine, norepinephrine, 2-OH-E2, 4-OH-E2, catechin, epicatechin, quercetin, fisetin, AdoMet, AdoHcy, 1,4-dithiothreitol, and isopropylthio-β-D-galactoside were purchased from Sigma-Aldrich (St. Louis, MO). [Methyl-3H]AdoMet (specific activity ) 11.2–13.5 Ci/mmol) was obtained from DuPont New England Nuclear Research Products (Boston, MA). All solvents used in this study were of HPLC grade or better and were obtained from Fisher Scientific Co. (Springfield, NJ). Cloning of the Human S-COMT and MB-COMT cDNAs. The human liver cDNA library (obtained from Stratagene, La Jolla, CA) was used as the template for cloning the human S-COMT and MBCOMT cDNAs. For PCR, the 5′ complementary forward primers (5′-CAA CAT ATG CCG GAG GCC CCG-3′ and 5′-GCA TAT GCC GGA GGC CCC GCC TC-3′) were used for S-COMT and MB-COMT, respectively, along with a common 3′ reverse primer (5′-CAG GAT CCT CAG GGC CCT GCT-3′). These primers were specifically designed to append the sequence that contains a 5′ NdeI restriction site and a 3′ BamHI restriction site (Figure 2, lower

panel). The resulting 655-bp fragment for S-COMT and the 815bp fragment for MB-COMT amplified by PCR were eluted using a Gel Extract Kit (Qiagen, Valenica, CA), and they were then ligated to the pGEM T vector (Promega, Madison, WI) using T4 ligase (Invitrogen, Carlsbad, CA). The products of the ligation reactions were transformed into the chemically competent E. coli TOP-10F′ cells (Invitrogen, Carlsbad, CA), and the transformed bacteria were then selected with ampicillin (50 µg/mL) on LB agar plates. The plasmids were purified using a Miniprep purification kit (Qiagen, Valencia, CA). The entire S-COMT and MB-COMT cDNA sequences were determined for verification. The plasmids were restriction-digested with NdeI and BamHI and ligated into the NdeI and BamHI-digested pET12a vector (Novagen, Madison, WI). The recombinant DNAs were introduced into chemically competent E. coli BL21 (DE3) (Novagen, Madison, WI) according to the procedures recommended by the manufacturer, and the transformed cells were selected with ampicillin (50 µg/mL) on LB agar plates. Bacterial Expression of Recombinant Human COMTs. For expression of the recombinant human S- and MB-COMT proteins in E. coli BL21 (DE3, expressing T7 polymerase), a positive clone was first cultured overnight at 37 °C in the LB medium supplemented with ampicillin (50 µg/mL). The culture broth was then inoculated into 300 mL fresh LB medium supplemented with ampicillin and incubated at 37 °C with vigorous shaking until the optical density reading of the bacterial culture mixture reached ∼0.6 (at λ ) 600 nm). The culture was then induced with isopropylthioβ-D-galactoside (at a final concentration of 0.5 mM) and cultured for another 3 h. The cells were collected by centrifugation and were then sonicated in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5 + 200 mM NaCl). After the addition of 5 mM 1,4-dithiothreitol and 1 mM phenylmethylsulfonyl fluoride (PMSF) to the crude homo-

1412 Chem. Res. Toxicol., Vol. 20, No. 10, 2007 genates, they were centrifuged at 10,000g for 10 min at 4 °C. The supernatants were then subjected to column purification or directly stored at -80 °C. Western Blot Analysis. The recombinant S- and MB-COMT proteins were analyzed using 12% SDS-polyacrylamide gel (SDS–PAGE) in a Mini-Protein system (BioRad, Hercules, CA). After electrophoresis, one of the gels was stained with Coomassie brilliant blue R250 to visualize the protein bands, and the protein bands on the other gel were transferred onto the PVDF membrane (BioRad, Hercules, CA) for Western blot analysis. The membrane was first blocked with 5% nonfat dried milk powder in Tris-buffered saline containing 0.1% Tween-20 (the blocking solution), and then it was probed with polyclonal rabbit antibodies (Chemicon, Temecula, CA) against the human COMT. The primary antibody–antigen complexes were detected using the donkey antirabbit IgG conjugated to horseradish peroxidase (GE Healthcare, Piscataway, NJ) and developed according to procedures supplied by Amersham ECL Plus (Piscataway, NJ). O-Methylation of Various Catecholic Substrates by Recombinant Human COMTs in Vitro. Several classes of representative endogenous and exogenous substrates were tested in the present study, which included catecholamines (dopamine, epinephrine, and norepinephrine), catechol estrogens (2-OH-E2 and 4-OH-E2), tea catechins (epicatechin and catechin), and bioflavonoids (quercetin and fisetin). For the in Vitro methylation of each of the substrates, the reaction mixture consisted of the recombinant COMT protein (at 16.2 µg/mL for S-COMT or 17.1 µg/mL for MB-COMT), 1.2 mM MgCl2, 100 µM AdoMet (containing 0.5–1 mCi [methyl-3H]AdoMet), 1 mM 1,4-dithiothreitol, and a catechol substrate (at indicated concentrations) in Tris-HCl buffer (50 mM, pH 7.4). The final volume of the reaction mixture was usually 300 µL. The reaction was initiated by the addition of the recombinant human COMT protein and carried out at 37 °C for 15–30 min. For the detection of the radioactive methylated products formed from different substrates, different methods had to be used depending on the substrate. For catecholamines, the reaction was stopped by the addition of 25 µL of 6 N HCl. After the removal of protein precipitates by centrifugation, the supernatant was analyzed for O-methylated products by using HPLC with a Spherisorb ODS column (5 µm particle size, 250 mm × 4.6 mm i.d.). The HPLC system consisited of a Waters 600E solvent gradient programmer, a Waters Lambda-Max model 481 UV detector, and a radioactive flow detector (β-RAM from IN/US). The solvent system consisited of a mixture of acetonitrile (solvent A), double-distilled water (solvent B), and 100 mM ammonium formate at pH 3 (solvent C). The optimized solvent mixtures (A/B/C) for the elution of dopamine, norepinephrine and epinephrine were isocratic at 5/85/10, 0/90/10, and 2/88/10, respectively. For catechol estrogens (2-OH-E2 and 4-OH-E2), the reaction was arrested by immediately placing the tubes on ice, followed by the addition of 500 µL ice-cold saline. The reaction mixtures were extracted with 5 mL ethyl acetate for the methylated catechol products. After centrifugation at 1000g for 10 min, the organic extracts were transferred to another set of tubes and dried under a stream of nitrogen. The methylated products of catechol estrogens were determined by using an HPLC method as described earlier (26). For bioflavonoids and tea catechins, the extraction was carried out by using ethyl acetate. After centrifugation at 1000g for 10 min, portions of the organic extracts were measured for radioactivity content with a liquid scintillation analyzer (Packard Tri-CARB 2900TR; Downers Grove, IL) as described in our earlier study (27). The rate of methylation of each substrate was expressed as nmol of methylated product formed/mg of human COMT protein/min (abbreviated as nmol/mg protein/min). The kinetic parameters (KM and VMAX values) were calculated by using the curve regression method of the SigmaPlot program. Development of Methods for Computational Modeling Analyses of Human COMTs. Energy minimizations, molecular dynamics (MD) simulations and molecular docking experiments were performed on a Silicon Graphics Origin 350 workstation using the extensible systematic force field (ESFF) (28), implemented in the InsightII modeling program (Version 2005, Accelrys Inc. San

Bai et al. Diego, CA). For energy minimizations, the steepest descent method was employed first to a 1000 kcal/molÅ energy gradient and followed by the Polak and Ribiere conjugate gradients method (29) until the final convergence criterion reached the 0.01 kcal/molÅ energy gradient. The atom-based method with the cut-off of 12.0 Å and the distance-dependent dielectric constant (ε ) εor, with εo ) 4.0) was used for the summation of the nonbonding interactions, unless otherwise specified. Construction of the Homology Model of Human S-COMT. First, sequence analysis of the human S- and MB-COMTs and their alignment with the rat S-COMT using the CLUSTAL W method (30) revealed over 80% sequence homology (see Supporting Information, Figure S1). On the basis of the earlier suggestion that the Class I AdoMet-dependent methyl transferases share similar catalytic core regions (31–33), a homology model of the human S-COMT was thus constructed according to the X-ray structure of the rat liver S-COMT (PDB code: 1VID), which served as a template (32). AdoMet, Mg2+ ion, and the crystallographic water that coordinates with Mg2+ (32) were also included. The protonation state of the side-chain nitrogen atom of K144/1942 was assigned to be neutral assuming that this nitrogen would play a key role in the catalytic process as the H-bond acceptor when it forms an H bond with the hydroxyl group of the substrate that is to be methylated (34, 35). The whole enzyme structure was then subjected to a 500-ps MD simulation. During the simulation, the highly conserved eight R-helical and seven β-sheet structures as previously identified in the rat COMT X-ray structure (32) were constrained along with the catalytic site residues important for catalytic activity (namely, D141/191, K144/194, D169/219, N170/220, and E199/ 249), the crystallographic water molecule, and Mg2+ ion. A structure was collected every 1 ps, and an average structure over the last 100 conformations was obtained and then subjected to energy minimization. Construction of the Homology Model of Human MBCOMT. Compared to S-COMT, human MB-COMT contains 50 additional amino acid residues at its N-terminus. Given the fact that the O-methylation of the catecholamine substrates by MBCOMT had markedly lower KM values than S-COMT (described in the Results section), we hypothesized that MB-COMT may contain N-terminal residues critical for substrate binding. In the present study, we included residues W38 through D53 to build the human MB-COMT model, while the N-terminal anchor residues (M1 through G37) were excluded because these residues did not appear to be directly involved in substrate-binding interactions. The secondary structure of these N-terminal residues (i.e., residues W38 through P45) of human MB-COMT was predicted to be R-helical (see Supporting Information). The human MB-COMT model was constructed in two steps: (i) Determination of the position of the R-helix (W38 through P45) in MB-COMT. Note that for the homology model of MB-COMT, the section that has the same amino acid sequence as that of S-COMT was adopted from the homology model built for S-COMT. (ii) Determination of the loop conformation that connects the R-helix and the section that has the same amino acid sequence as that of S-COMT. As a general strategy, a number of MB-COMT models with different N-terminal R-helix orientations were built by repeating the above-described procedures, and each of them was systematically evaluated to select the best-fitted models by determining the binding energy values of representative substrates in two different conformations. Specifically, the ZDOCK protein–protein docking program (36) was employed to determine the orientation of this R-helical patch (i.e., W38 through P45) to S-COMT. The best orientation of the helical patch was selected among the top ZDOCK candidates with high docking scores such that the patch is positioned on top of the active site. The loop structure of I46 through D53 was determined using the loop search method in the Homology module of the 2 The two numbers used here as well as in other places of this paper refer to the amino acid numbers of the human S-COMT and MB-COMT, respectively.

Kinetic and Molecular Studies of Human COMTs InsightII program. The resulting whole enzyme structure was then subjected to a 500 ps MD at 300 K molecular dynamics simulation. During the simulation, only the side chains of the residues W38 through D53 within 10 Å reach were allowed to move freely, and the R-helical structure of W38 through P45 was kept by constraining the backbone H-bonding. The catalytic site residues important for catalytic activity (namely, D141/191, K145/194, D169/219, N170/ 220, and E199/249), the crystallographic water molecule, and the Mg2+ ion were also constrained. A structure was collected every 1 ps, and an average structure over the last 100 conformations was obtained. The obtained structures were then subjected to energy minimization under the same conditions. The validity of the final structure was tested by using PROSTAT implemented in InsightII. Methods for Molecular Docking of Substrates into the Active Site of COMTs. An energy-minimized substrate structure was constructed using the Builder module in the InsightII program. In the database, the X-ray structure of the rat liver COMT (PDB code: 1VID) (32) was deposited as a complex structure with the substrate 3,5-dinitrocatechol at the active site. Therefore, after the human S- and MB-COMT structural models were superimposed to the X-ray structure of rat S-COMT, the catechol moiety of each target substrate was positioned to the catechol moiety of the substrate in the rat S-COMT X-ray structure. The binding pocket was defined to include all amino acid residues within 6 Å reach of the substrate. The simulated annealing method was performed to explore the binding conformations of the substrate using the following steps as a cycle: (i) MD simulations were performed by heating the system to 1500 K in 15 ps. (ii) The system was cooled down to 300 K in 30 ps. (iii) The conjugate gradient method was used to minimize the resulting structure. During the search, the catechol ring moiety of the substrate was held fixed by Mg2+ coordination while the defined binding site residues were allowed to move freely. The key components forming the catalytic site (the key binding site residues mentioned above, AdoMet, Mg2+, and water) were also held fixed during the simulation. A set of 100 conformations were collected by repeating this cycle and subjected to energy minimizations with the backbone of the structurally conserved R-helical and β-sheet regions and the catalytic site residues weakly constrained (force constant k ) 5.0 kcal/Å). The docking conformation with the lowest binding energy was selected as the likely binding conformation. Methods for Determining the LIE Energy for Substrates and COMTs. The enzyme–substrate binding free energy (∆Gbinding) was approximated by employing the linear interaction energy (LIE) method (37). In this method, ∆Gbinding ) R 〈∆Uvdw〉 + β 〈∆Uelec〉 + γ ∆SASA, where 〈∆Uvdw〉 or 〈Uelec〉 denotes the average change in the van der Waals or the electrostatic interaction energy of the ligand in the free and bound states, respectively, and ∆SASA is the change in the solvent-accessible surface area (SASA). The R, β, and γ are adjustable parameters that would best fit the experimental binding free energy data. In brief, to sample the enzyme–substrate complex that was required for determining the LIE energy ∆Gbinding, a series of hybrid Monte Carlo (HMC) simulations at 300 K were carried out. The system was initially heated to 300 K in 20 ps and then subjected to a MD simulation for 50 ps. A residue-based cutoff of 15 Å was set for the nonbonding interactions. The nonbonded pair list was updated every 10 fs. The time integration step of 1.0 fs and sampling LIE energies every 10 steps was used. During the MD simulations, all of the residues of the protein beyond 20 Å from the bound ligand, the catalytic residues coordinating to Mg2+, or the catechol moiety of the substrate were frozen. Similarly, the average LIE energies for the substrate will be obtained by performing MD simulations, in the same way as that for the enzyme–substrate complex. The SASA term required for predicting ∆Gbinding was obtained from the cavity term of the implemented Surface Generalized Born continuum solvation model (38).

Results Cloning and Selective Expression of Recombinant Human COMTs in E. coli. With human liver cDNAs as the

Chem. Res. Toxicol., Vol. 20, No. 10, 2007 1413

Figure 3. (A) Expression of human recombinant S- and MB-COMT proteins. Lane 1: molecular markers (192, 117, 99, 54, 27, 21, and 15 kDa). Lane 2: transformed with pET12a vector only. Lanes 3 and 4: S-COMT and MB-COMT, respectively. Samples were subjected to SDS–PAGE (12% gel) and visualized after staining with Coomassie blue. (B) Western blot analysis of the expression of S- and MB-COMTs. The expressed S- and MB-COMT proteins were resolved in the SDS–PAGE and then transferred to a PVDF membrane, and the Western blot analysis was performed using the polyclonal antihuman COMT antibody. COMT proteins were detected with a second antibody conjugated with horseradish peroxidase. Lane 5: Transformed with pET12a vector only. Lane 6 and 7: S-COMT and MB-COMT, respectively.

template, two forms of the human COMT gene were selectively amplified using PCR. After the recombinant plasmids containing the cDNA for human S-COMT or MB-COMT were enzymatically digested, gel electrophoresis of the digests revealed that the DNA fragment bands matched the expected sizes of 655 and 815 bp, respectively. The full-length DNA sequences were determined twice and compared with the known human COMT gene sequences. We found that the S-COMT and MB-COMT cDNAs cloned in this study encode Val108 in S-COMT and Val158 in MB-COMT, which means that they are the usual thermostable form of the enzymes (39, 40). After transformation of the E. coli BL21 (DE3) with the expression vector pET12a containing the gene for human S-COMT or MB-COMT, the bacteria that expressed the desired protein products were harvested, and the supernatants from the lysed bacteria were analyzed by 12% SDS–PAGE for Western blotting. Two predominant bands with apparent molecular masses of approximately 24 and 30 kD were detected, which matched the expected sizes for the recombinant human S- and MB-COMT proteins, respectively (Figure 3). Further column purification of the S- and MB-COMT proteins yielded highly pure proteins with the same molecular sizes (data not shown). Basic Biochemical Properties of Recombinant Human COMTs. The recombinant human S- and MB-COMT proteins were then used to study their biochemical kinetic properties for the O-methylation of various endogenous and exogenous catechol substrates. Using 2-OH-E2 and 4-OH-E2 as representative substrates, we first determined the effect of incubation time and COMT protein concentrations on the O-methylation of the catechol-E2 substrates (Figure 4A and B). On the basis of these data, an incubation time of 15 min and a protein concentration of 16.2 µg/mL for S-COMT or 17.1 µg/mL for MB-COMT were used in all other experiments so that the in Vitro methylation reactions proceeded within the optimized linear ranges. The apparent KM value of S-COMT for AdoMet was 113.6 or 145.3 µM, respectively, when 2-OH-E2 or 4-OH-E2 (at 10 µM) was used as substrate (Figure 4C). However, with these two substrates, the apparent KM values of MB-COMT for AdoMet were only 14.4 and 5.8 µM, which were 8 to 25 times lower than the corresponding KM values of S-COMT.

1414 Chem. Res. Toxicol., Vol. 20, No. 10, 2007

Bai et al.

Figure 4. Biochemical properties of the in Vitro O-methylation of catechol substrates with respect to incubation time (A), protein concentrations (B), AdoMet concentrations (C), AdoHcy concentrations (D), incubation pH (E), Mg2+ concentrations (F), and thermostability (G). The representative substrates used in these assays were 2-OH-E2 and 4-OH-E2 at 10 µM concentration. The incubation mixture consisted of the substrate, 100 µM [methyl-3H]AdoMet (containing 0.2 µCi (or as indicated), 16.2 µg/mL of S-COMT or 17.1 µg/mL of MB-COMT protein (or as indicated), 1 mM 1,4-dithiothreitol, and 1.2 mM MgCl2 (or as indicated) in a final volume of 300 µL Tris-HCl buffer (50 mM) at pH 7.4 or as indicated. The incubations were carried out at 37 °C for 15 min or as indicated. To test the thermostability of the S- and MB-COMTs, the enzymes were first incubated at 37 °C for the indicated length of time immediately before testing their catalytic activity for the O-methylation of 2-OH-E2 (at 10 µM). Each value is the mean of duplicate measurements.

Although S- and MB-COMTs have very different binding affinities for AdoMet, and the inhibition potency of AdoHcy (the demethylated AdoMet) for the O-methylation of 4-OH-E2 by the two COMTs was quite comparable (IC50 of 22.6 and 19.2 µM, respectively; Figure 4D). However, AdoHcy had a nearly 3-fold higher potency for inhibiting the methylation of 2-OH-E2 by S-COMT than that by MB-COMT (Figure 4D). The methylation of 2-OH-E2 and 4-OH-E2 by S-COMT or MB-COMT was pH-dependent. The overall pattern of pH dependence was similar for both S- and MB-COMTs, with peak catalytic activity seen at pH 7 to 8 (Figure 4E). It is known that the catalytic activity of COMT for the O-methylation of catechol substrates required the presence of Mg2+ as a catalytic cofactor. When 10 µM 2-OH-E2 was used as a representative substrate, the apparent KM values of Mg2+

for S- and MB-COMTs were nearly identical, at approximately 2 mM (Figure 4F). A stability test showed that the recombinant human S- and MB-COMT proteins were quite stable at 37 °C (Figure 4G). Notably, when two experimentally created mutant forms of the recombinant human S- and MB-COMTs were tested in the present study under the same conditions, they lost 20–50% of catalytic activity after preincubation at 37 °C for 2 h (data not shown). Lastly, it is of note that when 2-OH-E2 and 4-OH-E2 were used as substrates, we observed that the human S-COMT methylated 2-OH-E2 roughly twice as fast as it methylated 4-OH-E2, while MB-COMT methylated both catechol-E2 substrates at comparable rates (Figure 4). This pattern was consistently observed under various reaction conditions, such as at different incubation times,

Kinetic and Molecular Studies of Human COMTs

Chem. Res. Toxicol., Vol. 20, No. 10, 2007 1415

Figure 5. Relationship between catecholamine concentrations and their rate of methylation by S- and MB-COMTs. Three catecholamines were assayed as substrates: dopamine (upper panels), epinephrine (middle panels), and norepinephrine (lower panels). Note that for each catecholamine substrate, the middle and right panels showed the Michaelis–Menten curves for the COMT-mediated O-methylation at its C-3 (meta) and C-4 (para) hydroxy groups separately, whereas the left panel showed the curves for the combined total methylation (meta + para). The incubation mixture consisted of nine different concentrations (0, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 µM) of each substrate, 100 µM [methyl3 H]AdoMet, 16.2 µg/mL of S-COMT or 17.1 µg/mL of MB-COMT protein, 1 mM 1,4-dithiothreitol, and 1.2 mM MgCl2 in a final volume of 300 µL of Tris-HCl buffer (50 mM) at pH 7.4. The incubations were carried out at 37 °C for 30 min. Each value is the mean of duplicate measurements.

in the presence of different concentrations of COMT or AdoMet, or at different reaction pH values. Kinetic Parameters of the Recombinant Human COMTs for Various Catechol Substrates. The kinetic parameters were determined under optimized reaction conditions where the incubation time and enzyme concentrations were within the linear range, the AdoMet concentration was 100 µM, and the final pH of the mixture was 7.4. 1. Catecholamines (Dopamine, Epinephrine and Norepinephrine). The kinetic parameters (KM and VMAX) for each of the catecholamines were determined (Figure 5), and each value listed in Table 1 is the mean of two separate experiments. The apparent KM values for the O-methylation of catecholamines by MB-COMT differed markedly from the corresponding KM values for S-COMT. Using dopamine as an example, MB-COMT had a 16-fold higher affinity (1/KM) for its O-methylation than did S-COMT. In addition, the kcat values for MB-COMT-mediated 3-O-methylation of catecholamines were 2–4 times higher than those for S-COMT (Table 1).

To determine the regioselectivity of the methylation at the 3-hydroxyl (meta) and 4-hydroxyl (para) positions of catecholamines by S- and MB-COMTs, we separately quantified the 3-Oand 4-O-methylation products. The Michaelis–Menten curves are shown in Figure 5, and the KM and VMAX values are summarized in Table 1. It is apparent that the MB-COMT had a markedly higher binding affinity (i.e., lower KM values) and also higher VMAX values for the 3-O-methylation of catecholamines than for their 4-O-methylation. This finding agrees perfectly with the knowledge that catecholamine 3-O-methyl ethers are the most predominant metabolites present in ViVo. In comparison, this regioselectivity was relatively less pronounced with S-COMT. S-COMT had higher VMAX values for the 3-Omethylation of catecholamines than for their 4-O-methylation, but it had slightly higher KM values (i.e., lower affinity) for catalyzing the 3-O-methylation than 4-O-methylation (Table 1). 2. Catechol Estrogens (2-OH-E2 and 4-OH-E2). The KM values of MB-COMT for 2-OH-E2 and 4-OH-E2 were similar to the KM values of S-COMT for these two substrates (Figure

µM µM µM µM µM µM µM

100 250 250 250 100 250 100

AdoMet (0–400 µM) AdoMet (0–400 µM)

4-OH-E2 (0–80 µM) catechin (0–50 µM) epicatechin (0–50 µM) quercetin (0–50 µM)

2-OH-E2 (10 µM) c 4-OH-E2(10 µM) c c

c

total methylation total methylation

total methylation 3-O-methylation 4-O-methylation total methylation 3-O-methylation 4-O-methylation total methylation 3-O-methylation 4-O-methylation total methylation 2-O-methylation 3-O-methylation total methylation total methylation total methylation total methylation total methylation total methylation total methylation

methylation reaction measured

113.6d 145.3d

263.2 267.5 246.2 394.7 406.8 311.8 224.1 246.9 124.6 3.6 4.6 4.1 4.5 8.9 25.7 1.5 1.6 1.5 1.3

S-COMT

18.3d 17.1d

17.0 15.7 46.0 28.8 27.9 59.2 30.6 29.6 68.2 3.2 3.9 5.0 6.4 2.2 4.3 ND ND ND ND

MB-COMT

14.4d 5.8d

8.7 7.0 1.7 8.4 7.5 0.9 4.1 3.8 0.4 9.7 6.1 3.8 3.5 17.8 16.7 6.9 ND 11.1 ND

S-COMT

30.1d 25.9d

36.1 32.9 3.5 46.3 44.0 2.4 33.3 31.5 1.9 16.3 10.8 3.8 14.3 47.9 58.8 ND ND ND ND

MB-COMT

VMAX (nmol/mg/min)

ND ND

5.3 4.3 1.0 5.1 4.6 0.6 2.5 2.3 0.2 5.9 3.7 2.0 2.1 10.9 10.2 4.2 ND 6.9 ND

S-COMT

ND ND

11.6 10.6 1.1 14.9 14.1 0.8 10.7 10.1 0.6 5.1 3.5 1.1 4.6 15.4 18.9 ND ND ND ND

MB-COMT

kcat (min-1)

a Note: The concentrations of AdoMet used were indicated in the table. ND, not determined. b The rate of its total methylation was based on liquid scintillation counting of the total radioactivity extracted with ethyl acetate. The rates for its 2-O- and 3-O-methylation were determined by using HPLC that separately quantified the amount of 2-methoxyestradiol and 2-OH-E2 3-methyl ether formed. c To determine the apparent KM values of AdoMet for S-COMT and MB-COMT, a fixed concentration (at 10 µM) of 2-OH-E2 or 4-OH-E2 was used as substrate, and different concentrations of AdoMed (at 0–400 µM) were used. d The apparent KM and VMAX values were determined for AdoMet when using a fixed concentration of 2-OH-E2 or 4-OH-E2 as substrate.

fisetin (0–50 µM)

2-OH-E2 (0–80 µM)

100 µM

100 µM

norepinephrine (0–400 µM)

b

100 µM

epinephrine (0–400 µM)

AdoMet

100 µM

substrate (concentrations)

dopamine (0–400 µM)

KM (µM)

Table 1. Kinetic Parameters for the O-Methylation of Various Substrates Catalyzed by the Recombinant Human S-COMT and MB-COMTa

1416 Chem. Res. Toxicol., Vol. 20, No. 10, 2007 Bai et al.

Kinetic and Molecular Studies of Human COMTs

Figure 6. Relationship between catechol estrogen concentrations and their rate of methylation by S- and MB-COMTs. The upper panels showed the rate of total methylation of 2-OH-E2 and 4-OH-E2, and the lower panels showed the rate of 3-O-methylation and 4-Omethylation of 2-OH-E2 separately. The incubation mixture consisted of 10 different concentrations (0, 0.31, 0.63, 1.25, 2.5, 5, 10, 20, 40, and 80 µM) of each substrate, 100 µM [methyl-3H]AdoMet, 16.2 µg/mL of S-COMT or 17.1 µg/mL of MB-COMT protein, 1 mM 1,4-dithiothreitol, and 1.2 mM MgCl2 in a final volume of 300 µL of Tris-HCl buffer (50 mM) at pH 7.4. The incubations were carried out at 37 °C for 15 min. Note that the rate of its total methylation was based on liquid scintillation counting of the total radioactivity extracted with ethyl acetate. The rates for its 2-O- and 3-O-methylation were determined by using HPLC that separately quantified the amount of 2-methoxyestradiol and 2-OH-E2 3-methyl ether formed. Each value is the mean of duplicate measurements.

6). Notably, there was a weak self-inhibition of the catalytic activity by each of the two substrates at higher substrate concentrations (40–80 µM). While the kcat values for MBCOMT-mediated methylation of 2-OH-E2 and 4-OH-E2 were similar, the kcat value for S-COMT-mediated methylation of 2-OH-E2 was approximately twice as high as that for 4-OHE2. When the MB-COMT and S-COMT were compared against each other, the MB-COMT had a kcat value for the Omethylation of 2-OH-E2 approximately 50% higher than the kcat value of S-COMT, and the former had approximately 3-fold higher kcat value for the methylation of 4-OH-E2 than the latter. Using 2-OH-E2 as a representative catechol-E2 substrate, we have also separately quantified the formation of each of the mono-methyl ethers to probe the regioselectivity of its methylation catalyzed by S- and MB-COMTs (Figure 6). While Sand MB-COMTs had comparable KM values for the 2-O- and 3-O-methylation of 2-OH-E2, the difference in their VMAX values was evident, which favors the 2-O-methylation of 2-OH-E2, and thus, 2-methoxyestradiol was the major product. It is of note that MB-COMT had a more pronounced difference in the VMAX value favoring 2-O-methylation than did S-COMT. This in Vitro regioselectivity agreed well with the earlier observation that 2-methoxyestradiol is the quantitatively predominant metabolite of 2-OH-E2 formed in ViVo (41).

Chem. Res. Toxicol., Vol. 20, No. 10, 2007 1417

Figure 7. Relationship between dietary catechol concentrations and their rate of methylation by S- and MB-COMTs. The incubation mixture consisted of 9 different concentrations (0, 0.39, 0.78, 1.57, 3.13, 6.25, 12.5, 25, and 50 µM) of each substrate, 250 µM [methyl-3H]AdoMet, 16.2 µg/mL of S-COMT or 17.1 µg/mL of MB-COMT protein, 1 mM 1,4-dithiothreitol, and 1.2 mM MgCl2 in a final volume of 300 µL of Tris-HCl buffer (50 mM) at pH 7.4. The incubations were carried out at 37 °C for 15 min. Each value is the mean of duplicate measurements.

3. Catecholic Dietary Polyphenols (Quercetin, Fisetin, Catechin, and Epicatechin). We also determined the kinetic parameters (KM and VMAX) when representative dietary polyphenolic substrates were used as substrates. The O-methylation of catechin and epicatechin (two representative tea catechins) by recombinant human S-COMT followed typical Michaelis– Menten curve patterns (Figure 7). The O-methylation of these two tea catechins by MB-COMT basically had similar curve patterns, except there was a weak self-inhibition of the catalytic activity by catechin (but not by epicatechin) at concentrations >12 µM. The apparent KM values for the O-methylation of catechin and epicatechin by S-COMT were ∼4 times higher than the KM values for MB-COMT-mediated methylation (Figure 7, Table 1), suggesting that the MB-COMT had a higher binding affinity for these substrates than S-COMT. Also, MBCOMT had slightly higher kcat values for the methylation of these substrates. The O-methylation of quercetin and fisetin catalyzed by S-COMT roughly followed the Michaelis–Menten curve patterns but with a weak self-inhibition at higher substrate concentrations (>12.5 µM) (Figure 7). The apparent KM and VMAX values were determined according to the regression analysis of the Michaelis– Menten curves at lower substrate concentrations (at 0–12.5 µM). However, the O-methylation of quercetin and fisetin by MBCOMT exhibited an unusual curve pattern, with an abrupt and strong substrate concentration-dependent self-inhibition (Figure 7). The kinetic parameters could not be meaningfully assessed. Notably, the distinct curve patterns for the O-methylation of quercetin and fisetin by S-COMT and MB-COMT were not affected when different concentrations (100 and 250 µM) of AdoMet were used. Computational Molecular Modeling Studies. 1. Human COMT Homology Structure Models. Sequence alignment analysis of rat S-COMT with human S-COMT showed over

1418 Chem. Res. Toxicol., Vol. 20, No. 10, 2007

Figure 8. Homology modeled 3-D structure of human MB-COMT in ribbon representation colored by the secondary structural elements (Rhelix, orange; β-sheet, blue; and turn and random coil, green). AdoMet (hydrogen atoms omitted) and the crystallographic water are represented in stick depiction and Mg2+ in CPK depiction. The core fold is shown incorporating alternative R-helices (RA-RE, and RZ) and β-sheets (β1β7) in the order of RZ-β1-RA-β2-RB-β3-(RC)-β4-RD-β5-RE-β6-β7. It should be noted that RC is not conserved. The numbering is adopted from a recent paper (34). The N-terminal 16 amino acid residues extended from the S-COMT (refer to text) are colored red. Color coding: white, C; red, O; blue, N; yellow, S; magenta, Mg2+; and cyan, H.

80% sequence identity. Because of the high sequence homology, the structurally conserved regions in the rat S-COMT X-ray structure are assumed to be also conserved in the human COMT. In the computational models developed in the present study, the structurally conserved R-helical and β-sheet regions were largely constrained. According to the homology models for the human S- and MB-COMTs developed in this study, it appears that the overall binding pocket structures and conformations of these two COMTs are similar, and each has a rather shallow groove on the enzyme surface, which can readily accommodate substrates with different sizes and structures (the structure of MB-COMT is shown in Figure 8). The only apparent difference between these two enzymes is that the MB-COMT has an extension in the N-terminus, and its residues W38 through D53 (an R-helical patch) are positioned right on top of the substrate-binding pocket. It is of note that in this computational model, the additional N-terminal tail (in the case of MB-COMT) as well as the side walls of the binding groove (namely, the edge regions of β6 and β7 sheets) would interact with different substrates in different ways, which partially determines the binding affinity of the substrates and also their regioselectivity of methylation (more details are discussed below). 2. Substrate Docking Studies of Human COMTs. Using the homology models of human S- and MB-COMTs as described above, we determined the optimal docking conformations for each of the representative substrates, and we have also calculated their corresponding binding energy values (∆Gbinding). Representative data are summarized in Figure 9 and Table 2. In order to consider the solvation and entropy effects, we chose to estimate the ∆Gbinding values using the LIE method based on the Surface Generalized Born continuum solvation model (38). By fitting the calculated ∆Gbinding values from two regioselective conformations for each of the representative catechol substrates to their experimental ∆Gbinding values,

Bai et al.

we yielded the following LIE equation for MB-COMT: ∆Gbinding ) 0.415 〈∆Uvdw〉 + 0.012 〈∆Uelec〉 + 0.611 ∆SASA. The LIE equation for the S-COMT was ∆Gbinding ) 0.259 〈∆Uvdw〉 + 0.015 〈∆Uelec〉 – 1.047 ∆SASA. For both COMT forms, the root mean square deviation (rmsd) of the predicted ∆Gbinding values was