Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4.
Glutathione (GSH) exerted a profound effect on the oxidation of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and 7-benzyloxyquinoline (BQ) by human liver microsomes as well as by CYP3A4-containing insect cell microsomes (Baculosomes). The cooperativity in O-debenzylation of both substrates is eliminated in the presence of 1-4mM GSH. Addition of GSH also increased the amplitude of the 1-PB induced spin shift with purified CYP3A4 and abolished the cooperativity of 1-PB or BFC binding. Changes in fluorescence of 6-bromoacetyl-2-dimethylaminonaphthalene attached to the cysteine-depleted mutant CYP3A4(C58,C64) suggest a GSH-induced conformational changes in proximity of alpha-helix A. Importantly, the K(S) value for formation of the GSH complex and the concentrations in which GSH decreases CYP3A4 cooperativity are consistent with the physiological concentrations of GSH in hepatocytes. Therefore, the allosteric effect of GSH on CYP3A4 may play an important role in regulation of microsomal monooxygenase activity in vivo.
Davydov
Dmitri R
DR
Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1031, USA. d.davydov@utmb.edu
Davydova
Nadezhda Y
NY
Tsalkova
Tamara N
TN
Halpert
James R
JR
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000737:chemistry; D000495:Allosteric Site; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D001665:Binding Sites; D003374:Coumarins / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology; D009154:Mutation; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D011721:Pyrenes / Q000378:metabolism; D011804:Quinolines / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
C463480:1-pyrenebutanol; C122155:6-bromoacetyl-2-dimethylaminonaphthalene; C419147:7-benzyloxy-4-trifluoromethylcoumarin; D003374:Coumarins; D011721:Pyrenes; D011804:Quinolines; C065956:7-benzyloxyquinoline; D003577:Cytochrome P-450 Enzyme System; D015081:2-Naphthylamine; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D005978:Glutathione; D003545:Cysteine