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In this study, we characterized the enzymology of cytosolic catechol-O-methyltransferase (COMT)-catalyzed …


Biology Articles » Biochemistry » Enzymology » Enzymology of Methylation of Tea Catechins and Inhibition of Catechol-O-methyltransferase by ()-Epigallocatechin Gallate » Results

Results
- Enzymology of Methylation of Tea Catechins and Inhibition of Catechol-O-methyltransferase by ()-Epigallocatechin Gallate

Time Course of Methylation of EGCG, 4"-MeEGCG, and EGC by Rat Liver Cytosolic COMT. The possible sites of methylation and glucuronidation of EGCG and EGC, based on our present and previous studies, are summarized in Fig. 2. In the presence of SAM, EGCG (1 µM) was readily methylated to 4"-MeEGCG by rat liver cytosolic COMT (Fig. 3A). 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 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. 3B 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. 3C). 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 3D showed that EGCG-4"-Gluc was also methylated, although at a slightly slower rate than EGCG.

Concentration-dependent Methylation of EGCG and EGC in Humans, Rats, and Mice. 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. 4). The apparent kinetic parameters of methylation of EGCG and EGC by the liver cytosolic COMT of humans, mice, and rats are summarized in Table 1. In all three species, the Km values of EGCG for COMT were much lower than EGC and the catalytic efficiencies (Vmax/Km) for the methylation of EGCG were 3- to 9-fold higher than that for EGC. Rat liver had the highest Vmax values in methylating EGCG and EGC; whereas human liver had the highest Vmax/Km values for the methylation of EGCG and EGC.

Effect of Substrate Concentration on the Product Profile of EGCG Methylation. Previous in vivo studies suggest that the product profile of EGCG methylation is dose-dependent (Kida et al., 2000; Kohri et al., 2001; Meng et al., 2002). 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. 5A). Methylation of EGCG formed 4"-MeEGCG and 4',4"-DiMeEGCG, and they were plotted separately as percentages of their sum in Fig. 5B. 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. 5B).

Methylation of EGCG and EGC by Small Intestinal Cytosolic COMT. The methylation of EGCG by small intestinal cytosolic COMT (Fig. 6A) was approximately 10-fold slower than that by the liver cytosolic COMT (Fig. 5B). In contrast to the very low capacity of EGCG methylation (Fig. 6A), the activities of EGC methylation by small intestinal cytosolic COMT were much higher (Fig. 6B). Rats had much higher intestinal COMT activities than mice with either EGCG or EGC as the substrate.

Effect of Glucuronidation on Methylation of EGC and EGCG. 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.

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. 7A) had the same product-ion spectrum as the glucuronide of 4"-MeEGCG (Fig. 7B), 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. 7C). The product-ion m/z 485 (Fig. 7, D and E) indicated the formation of di-methylated products, and P1 and P2 were tentatively identified as 3',4'-di-O-methyl-EGCG-4"-Gluc and 3',5'-di-O-methyl-EGCG-4"-Gluc. The 3'- and 5'-positions are symmetrical.

 

Inhibition of COMT Activity by Catechins and Their Derivatives. In view of the low Km 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. 8A), with a similar IC50 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.

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. 2 showed COMT-inhibitory activities (Table 2). 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.

 

When L-DOPA was used as a substrate, EGCG and its derivatives showed similar potency as they inhibited the methylation of EGC (Table 2). The inhibitory activities of the four major catechins followed the order of EGCG > ECG > EGC > (-)-epicatechin. Methylation and glucuronidation of EGC on the B-ring greatly decreased their COMT-inhibiting activities.

 

Mechanism of Inhibition of COMT Activity by EGCG and Methylated EGCG. The inhibition of EGC methylation by EGCG was uncompetitive with respect to SAM (Fig. 8B), 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 Vmax (pmol/mg/min) and Km (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. 8C); with the increase of EGCG concentrations, the Vmax values for EGC methylation decreased, whereas the corresponding Km values increased. In the presence of different concentrations of 4"-MeEGCG (Fig. 8D) and 4',4"-DiMeEGCG (Fig. 8E), the Vmax values for EGC methylation decreased in a concentration-dependent manner, whereas the corresponding Km 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. 8, D and E). The Lineweaver-Burk plot (Fig. 8F) indicated that 4',4"-DiMeEGCG also inhibited the methylation of L-DOPA in a noncompetitive manner.



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