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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 » Discussion

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

Our current studies demonstrate the rapid O-methylation of EGCG and EGC by liver cytosolic COMT and the potent inhibition of cytosolic COMT by EGCG and its metabolites. The much lower Km and higher Vmax/Km values of EGCG methylation than EGC methylation (Table 1) suggest that EGCG is a better substrate for COMT than EGC. The O-methylation at the 4'-position of EGC was rapid, suggesting that the B-ring of catechins is a favorable site for the COMT. With EGCG, the methylation on the 4"-position was strongly favored over the 4'-position, suggesting a higher affinity of the D-ring than the B-ring to COMT. Zhu et al. (2000, 2001) reported that the methylation of EGCG by rat liver cytosolic COMT was considerably slower than that of EGC. This is probably due to the high concentrations of EGCG (10-100 µM) and long incubation time (20 min) used.

When longer incubation time (20 min) was used for the methylation of EGCG (Fig. 5), 4',4"-DiMeEGCG was the major product at low EGCG concentrations ( results that after oral administration of green tea, 4',4"-DiMeEGCG was the major metabolite of EGCG detected in the urine samples of humans, mice, and rats (Meng et al., 2002). After an i.v. injection of 4 mg of EGCG, 4',4"-DiMeEGCG was also the major metabolites excreted in the bile of rats (Kohri et al., 2001). At higher concentrations (>3 µM) of EGCG, 4"-MeEGCG was the predominant product (Fig. 5B). In vivo, when a high dose of EGCG (100 mg) was administered orally, 4"-MeEGCG was the major methylation product of EGCG excreted in the bile of rats (Kida et al., 2000).

In phase II metabolism in vivo, EGCG could be methylated first and then conjugated by glucuronidation or sulfation or be conjugated first and then methylated. The lower Km values (0.2-0.5 µM) for EGCG methylation (Table 1) than those for glucuronidation (> 30 µM) by human and rodent liver microsomes (Lu et al., 2003) suggest that with low concentrations of EGCG (e.g., after drinking green tea), methylation of EGCG would proceed to form 4'-MeEGCG and then 4',4"-DiMeEGCG, and this indeed has been observed in vivo (Kohri et al., 2001; Meng et al., 2002). At very high doses of EGCG, glucuronidation may become more prominent because the Vmax (Lu et al., 2003) is higher than the methylation reaction. For example, EGCG-4"-Gluc is the sole glucuronide formed in the mouse small intestine and the major glucuronide formed in the mouse liver (Lu et al., 2003). This 4"-glucuronide can be methylated on the B-ring (Fig. 7, C-E). Therefore, different mono- and di-methylated products would be formed. This prediction is consistent with the observation that after administration of a large quantity of EGCG to mice, four mono-methylated and four di-methylated EGCG were detected in the urine after hydrolysis by beta-glucuronidase and sulfatase; of which, three mono-methylated EGCG had similar peak heights (Meng et al., 2002). This metabolism profile is not expected if EGCG does not undergo conjugation first; in that case 4"-MeEGCG would have been the predominant metabolite produced.

Our studies show that EGCG potently inhibits the activities of COMT. The gallated catechins (EGCG and ECG) have 60-fold higher activities than nongallated catechins (EGC and (-)-epicatechin) at inhibiting COMT activity, suggesting the importance of the D-ring for the inhibitory activity. When the 4"-position of D-ring is glucuronidated, inhibitory activity also decreases markedly. The 2- to 3-fold differences between trihydroxyl-catechins (EGCG and EGC) and dihydroxyl-catechins (ECG and (-)-epicatechin) suggest that the B-ring also contributes significantly to the COMT-inhibiting activity of EGCG. The noncompetitive inhibition of COMT by 4"-Me-EGCG and 4',4"-DiMeEGCG suggests that they inhibit COMT by binding to sites other than the catechol binding site. It has been proposed that many SAM-dependent methyltransferases, including DNA methyltransferases and COMT, have a common catalytic domain structure (Cheng, 1995). It is possible that EGCG/methylated EGCG can bind to certain sites on the catalytic domain of DNA methyltransferase and inhibit its activity.

L-DOPA is the drug of first choice in the treatment of Parkinson's disease. Inhibition of the peripheral clearance of L-DOPA by COMT and dopa decarboxylase increases its entry to the brain and subsequent conversion to dopamine. Our study shows that EGCG potently inhibits the methylation of L-DOPA. The IC50 of 0.2 µM is lower than the peak human blood levels of EGCG after taking 800 mg of EGCG (~1 µM). EGCG, as a potent COMT inhibitor, a mild irreversible inhibitor of dopa decarboxylase (Bertoldi et al., 2001), a neuroprotective agent in animal and cell models of Parkinson's disease (Levites et al., 2002), and a possible brain-penetrating chemical (Suganuma et al., 1998), may have beneficial effects in patients with Parkinson's disease. Regular tea drinking has been reported to be a protective factor against Parkinson's disease (Chan et al., 1998; Checkoway et al., 2002).

The present study characterized the COMT-catalyzed methylation of EGCG and EGC in humans, mice, and rats. The methylation of EGCG is highly dose-dependent. Additionally, these catechins and their metabolites are potent inhibitors of COMT. Further studies on effects of tea catechins on the metabolism of catecholic hormones and their related disease are warranted. The potential interactions between EGCG and catecholic hormones or drugs should also be considered.

Acknowledgments

We thank Dr. Chi-Tang Ho for providing some of the reagents for this study, and Drs. Anthony Lu and Joshua Lambert for critical reading of this manuscript. The LC/MS analysis was conducted in the Analytical Center (directed by Dr. Brian Buckley) at the Environmental and Occupational Health Sciences Institute. The mouse liver tissue for the biosynthesis of EGC glucuronides was collected from an experiment conducted by Drs. Yaoping Lu and Allan H. Conney at Rutgers University.

Footnotes

Received October 15, 2002; accepted February 3, 2003.

This work was supported by National Institutes of Health Grants CA 56673 and CA88961.


Address correspondence to: Dr. Chung S. Yang, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854. E-mail: csyang@rci.rutgers.edu

Abbreviations

Abbreviations used are: EGCG, ()-epigallocatechin gallate; EGC, ()-epigallocatechin; ECG, ()-epicatechin gallate; EGCG-7-Gluc, ()-EGCG-7-O-glucuronide (similar abbreviations for other glucuronides); 4'-MeEGC, 4'-O-methyl-()-epigallocatechin; 4"-MeEGCG, 4"-O-methyl-()-epigallocatechin gallate; 4',4"-DiMeEGCG, 4',4"-di-O-methyl-epigallocatechin gallate; COMT, catechol-O-methyltransferase; SAM, S-adenosylmethionine; L-DOPA, 3,4-dihydroxy-L-phenylalanine; 3-MeDOPA, L-3-O-methyl-DOPA; UDPGA, UGP-glucuronic acid; LC/MS/MS, liquid chromatographic tandem mass spectrometric method; HPLC, high-performance liquid chromatography; EGCG-4"-Gluc, ()-EGCG-4"-O-glucuronide; EGCG-3"-Gluc, ()-EGCG-3"-O-glucuronide; EGCG-3'-Gluc, ()-EGCG-3'-O-glucuronide; EGC-3'-Gluc, ()-EGC-3'-O-glucuronide; EGC-7-Gluc, ()-EGC-7-O-glucuronide.



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