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<p><strong>Time Course of Methylation of EGCG, 4"-MeEGCG, and EGC by Rat Liver Cytosolic COMT.</strong> The possible sites of methylation and glucuronidation of EGCG and EGC, based on our present and previous studies, are summarized in Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F2">2</a>. In the presence of SAM, EGCG (1 µM) was readily methylated to 4"-MeEGCG by rat liver cytosolic COMT (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F3">3</a>A). The level of 4"-MeEGCG reached a maximum within 5 min and then started to decrease. The production of 4',4"-DiMeEGCG was low before 2.5<sup> </sup>min and increased markedly after 5 min. This result suggests a sequential methylation of EGCG to 4"-MeEGCG and then to 4',4"-DiMeEGCG. The results in Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F3">3</a>B indicated the second step is slower than the first step. The methylation of EGC was linear within 5 min at an initial rate similar to that of EGCG (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F3">3</a>C). At 10 min, the level of 4'-MeEGC reached a plateau, but the level of 4"-MeEGCG decreased due to its conversion to 4',4"-DiMeEGCG. Upon incubating EGCG, EGC, or EGCG-4"-Gluc with rat liver cytosol in the presence of SAM, the disappearance of the substrate corresponded to the formation of the methylated product. Figure <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F3">3</a>D showed that EGCG-4"-Gluc was also methylated, although at a slightly slower rate than EGCG. </p> <p><strong>Concentration-dependent Methylation of EGCG and EGC in Humans, Rats, and Mice.</strong> Methylation of EGCG and EGC resulted in the formation of 4"-MeEGCG and 4'-MeEGC as the major products in short incubation (2 or 5 min), respectively, and the product formation approximately followed Michaelis-Menten kinetics (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F4">4</a>). The apparent kinetic parameters of methylation of EGCG and EGC by the liver cytosolic COMT of humans, mice, and rats are summarized in Table <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#T1">1</a>. In all three species, the <em>K</em><sub>m</sub> values of EGCG for COMT were much lower than EGC and the catalytic efficiencies (<em>V</em><sub>max</sub>/<em>K</em><sub>m</sub>) for the methylation of EGCG were 3- to 9-fold higher than that for EGC. Rat liver had the highest <em>V</em><sub>max</sub> values in methylating EGCG and EGC; whereas human liver had the highest <em>V</em><sub>max</sub>/<em>K</em><sub>m</sub> values for the methylation of EGCG and EGC. </p> <p><strong>Effect of Substrate Concentration on the Product Profile of EGCG Methylation.</strong> Previous in vivo studies suggest that the product profile of EGCG methylation is dose-dependent (Kida et al., 2000<a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#B10"><img src="http://dmd.aspetjournals.org/icons/ref-arrow.gif" alt=""></a>; Kohri et al., 2001<a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#B11"><img src="http://dmd.aspetjournals.org/icons/ref-arrow.gif" alt=""></a>; Meng et al., 2002<a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#B19"><img src="http://dmd.aspetjournals.org/icons/ref-arrow.gif" alt=""></a>). To further investigate this phenomena, concentration-dependent methylation of EGCG and 4"-MeEGCG by rat liver cytosol was studied using a 20-min incubation. At low concentrations ( and EGCG proceeded at the same rate, but at high concentrations (> 3.0 µM) of substrate, EGCG was the preferred substrate (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F5">5</a>A). Methylation of EGCG formed 4"-MeEGCG and 4',4"-DiMeEGCG, and they were plotted separately as percentages of their sum in Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F5">5</a>B. 4',4"-DiMeEGCG was the predominant product in incubations with low concentrations ( was the predominant product in incubations with high concentrations (> 3.0 µM) of EGCG (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F5">5</a>B). </p> <p><strong>Methylation of EGCG and EGC by Small Intestinal Cytosolic COMT.</strong> The methylation of EGCG by small intestinal cytosolic COMT (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F6">6</a>A) was approximately 10-fold slower than that by the liver cytosolic COMT (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F5">5</a>B). In contrast to the very low capacity of EGCG methylation (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F6">6</a>A), the activities of EGC methylation by small intestinal cytosolic COMT were much higher (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F6">6</a>B). Rats had much higher intestinal COMT activities than mice with either EGCG or EGC as the substrate. </p> <p><strong>Effect of Glucuronidation on Methylation of EGC and EGCG.</strong> EGC-7-Gluc was readily methylated by rat liver COMT (data not shown). When EGC-7-Gluc and EGC (each at 10 µM) were coincubated with rat liver cytosol, the methylation of EGC-7-Gluc or EGC was each inhibited by 50%, indicating that glucuronidation of EGC on the A-ring has no significant effect on its interaction with COMT. After incubation of rat liver cytosol with 10 µM of EGC-3'-Gluc or EGC-7-Gluc, the peak height of methylated EGC-3'-Gluc was only 1% that of methylated EGC-7-Gluc (analyzed by LC/MS/MS), suggesting that methylation of the B-ring is inhibited by the glucuronidation at the 3'-position. </p> Similarly, the methylation of EGCG was not affected by glucuronidation at the A-ring, but significantly hindered by glucuronidation at the B-ring. For example, after incubation of EGCG-7-Gluc with rat liver cytosol and SAM for 20 min, large peaks of both mono- and di-methylated EGCG-7-Gluc were detected (by LC/MS/MS). Methylation of EGCG-3'-Gluc, however, resulted in the formation of a mono-methylated product as the major product and only a very small amount of a di-methylated product (data not shown). The mono-methylated product of EGCG-3'-Gluc (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F7">7</a>A) had the same product-ion spectrum as the glucuronide of 4"-MeEGCG (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F7">7</a>B), suggesting that the methylation of EGCG-3'-Gluc is on the 4"-position of the D-ring. With 4"-position glucuronidated in EGCG-4"-Gluc, the only catechol site for COMT is on the B-ring. Two clear peaks of methylated EGCG-4"-Gluc with retention times of 24.4 (P1) and 24.9 (P2) min were observed (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F7">7</a>C). The product-ion <em>m/z</em> 485 (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F7">7</a>, D and E) indicated the formation of di-methylated products, and P1 and P2 were tentatively identified as 3',4'-di-<em>O</em>-methyl-EGCG-4"-Gluc and 3',5'-di-<em>O</em>-methyl-EGCG-4"-Gluc. The 3'- and 5'-positions are symmetrical. <p> </p> <p><strong>Inhibition of COMT Activity by Catechins and Their Derivatives.</strong> In view of the low <em>K</em><sub>m</sub> value of EGCG methylation, we investigated possible inhibitory effects of EGCG on the activities of cytosolic COMT with EGC as a substrate. The hepatic activities of COMT of rats and mice were inhibited by EGCG (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F8">8</a>A), with a similar IC<sub>50</sub> of ~0.15 µM. Rat liver cytosol was used in all of the subsequent studies on the inhibition of COMT due to the abundance of enzyme. </p> To elucidate the structural basis for the inhibition of COMT by EGCG, the inhibitory activities of EGCG, EGCG metabolites, and EGCG analogs were investigated using EGC (10 µM) as a substrate. All the methylated EGCG and EGCG glucuronides in Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F2">2</a> showed COMT-inhibitory activities (Table <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#T2">2</a>). EGCG-7-Gluc was less effective than EGCG, and EGCG-4"-Gluc was only one tenth as effective. ECG, which has one less hydroxyl group than EGCG on the B-ring, also potently inhibited COMT; whereas pyrogallol, which only contains the 3,4,5-trihydroxyl moiety of EGCG, was much less potent. <p> </p> When L-DOPA was used as a substrate, EGCG and its derivatives showed similar potency as they inhibited the methylation of EGC (Table <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#T2">2</a>). The inhibitory activities of the four major catechins followed the order of EGCG > ECG > EGC > (<img alt="-" src="http://dmd.aspetjournals.org/math/12pt/normal/minus.gif">)-epicatechin. Methylation and glucuronidation of EGC on the B-ring greatly decreased their COMT-inhibiting activities. <p> </p> <p><strong>Mechanism of Inhibition of COMT Activity by EGCG and Methylated EGCG.</strong> The inhibition of EGC methylation by EGCG was uncompetitive with respect to SAM (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F8">8</a>B), suggesting that there is no direct interaction between EGCG and the SAM-binding site on the COMT. With a saturating concentration of SAM (200 µM), the <em>V</em><sub>max</sub> (pmol/mg/min) and <em>K</em><sub>m</sub> (µM) values for the methylation of EGC by rat liver cytosol were 2752 ± 76 and 12.8 ± 1.5, respectively; EGCG displayed a mixed-type inhibition of COMT activity with respect to EGC (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F8">8</a>C); with the increase of EGCG concentrations, the <em>V</em><sub>max</sub> values for EGC methylation decreased, whereas the corresponding <em>K</em><sub>m</sub> values increased. In the presence of different concentrations of 4"-MeEGCG (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F8">8</a>D) and 4',4"-DiMeEGCG (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F8">8</a>E), the <em>V</em><sub>max</sub> values for EGC methylation decreased in a concentration-dependent manner, whereas the corresponding <em>K</em><sub>m</sub> values were unchanged, indicating a noncompetitive mechanism of enzyme inhibition with respect to EGC. 4"-MeEGCG appeared to be more potent than 4',4"-DiMeEGCG in inhibiting COMT activity (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F8">8</a>, D and E). The Lineweaver-Burk plot (Fig. <a href="http://dmd.aspetjournals.org/cgi/content/full/31/5/572#F8">8</a>F) indicated that 4',4"-DiMeEGCG also inhibited the methylation of L-DOPA in a noncompetitive manner. </p> <br>
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