Kinetics of the inhibition of bovine liver dihydrofolate reductase by tea catechins: origin of slow-binding inhibition and pH studies.
Dihydrofolate reductase (DHFR) is the subject of intensive investigation since it appears to be the primary target enzyme for "antifolate" drugs, such as methotrexate and trimethoprim. Fluorescence quenching and stopped-flow fluorimetry show that the ester bond-containing tea polyphenols (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) are potent and specific inhibitors of DHFR with inhibition constants (K(I)) of 120 and 82 nM, respectively. Both tea compounds showed the characteristics of slow-binding inhibitors of bovine liver DHFR. In this work, we have determined a complete kinetic scheme to explain the slow-binding inhibition and the pH effects observed during the inhibition of bovine liver DHFR by these tea polyphenols. Experimental data, based on fluorimetric titrations, and transient phase and steady-state kinetic studies confirm that EGCG and ECG are competitive inhibitors with respect to 7,8-dihydrofolate, which bind preferentially to the free form of the enzyme. The origin of their slow-binding inhibition is proposed to be the formation of a slow dissociation ternary complex by the reaction of NADPH with the enzyme-inhibitor complex. The pH controls both the ionization of critical catalytic residues of the enzyme and the protonation state of the inhibitors. At acidic pH, EGCG and ECG are mainly present as protonated species, whereas near neutrality, they evolve toward deprotonated species due to ionization of the ester-bonded gallate moiety (pK = 7.8). Although DHFR exhibits different affinities for the protonated and deprotonated forms of EGCG and ECG, it appears that the ionization state of Glu-30 in DHFR is critical for its inhibition. The physiological implications of these pH dependencies are also discussed.
Navarro-Perán
Enma
E
Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular A, Facultad de Biología, Universidad de Murcia, Murcia, Spain.
Cabezas-Herrera
Juan
J
Hiner
Alexander N P
AN
Sadunishvili
Tinatin
T
García-Cánovas
Francisco
F
Rodríguez-López
José Neptuno
JN
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D028241:Camellia sinensis; D002392:Catechin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D005419:Flavonoids / Q000737:chemistry* / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010636:Phenols / Q000737:chemistry* / Q000378:metabolism; D059808:Polyphenols; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013662:Tea; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism
D005419:Flavonoids; D005493:Folic Acid Antagonists; D010636:Phenols; D059808:Polyphenols; D013662:Tea; D002392:Catechin; C062669:epicatechin gallate; C045651:epigallocatechin gallate; D013762:Tetrahydrofolate Dehydrogenase