Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

Abstract

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 1-4 mM 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 α-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.

Inhibition of Cytochrome P450 Enzymes

“…It has also been suggested that in solution the mercaptide radicals can easily dimerize to form the disulfide and thus dissociate from the complexes [76] Similar thiol-binding to ferric CYP3A4 complexes accounts for its functionally relevant interactions with glutathione (GSH) [80], an important intracellular γ-glutamylcysteine-glycine tripeptide that serves as the cofactor for various detoxifying enzymes (peroxidases, GSH-transferases), as well as a nucleophilic antioxidant that traps and thus detoxifies reactive O 2 species (ROS), including free radicals and peroxides, and reactive electrophilic metabolites Because of this very property, and the assumption that GSH was not only far too large, but also too hydrophilic a molecule to enter the lipophilic P450 active sites, it has been often used in the past as a diagnos-tic probe to trap reactive metabolites that escape the P450 active site, and thus as an indicator of chemical reactivity external to the P450 active site However, it appears that some quite large and promiscuous P450 active sites such as that of CYP3A4 (and possibly that of CYP2C8), can accommodate GSH, as determined by the telltale split Soret UV-Vis difference spectrum characteristic of thiol interactions with the sixth ligand of the P450 heme iron [80; K K Korsmeyer & M A Correia, unpublished observations, 1995] Furthermore, this GSH-CYP3A4 binding exhibits positive homotropic cooperativity (Hill equation exhibiting an S 50 of 86 mM and a Hill coefficient of 22), thereby revealing an additional allosteric effector site for GSH-binding within the CYP3A4 active site [80] At physiologically relevant GSH concentrations, such GSH-CYP3A4 binding disrupts the substrate homotropic cooperativity assayed via the CYP3A4-dependent O-debenzylation of 7-benzyloxy-4-(trifluoromethyl)-coumarin (7-BFC) and 7-benzyloxyquinoline, as well as that monitored through spectrally detectable substrate binding [80] However, not all substrate-effector interactions were similarly affected For instance, GSH increased CYP3A4 binding of 1-pyrenebutanol (1-PB) monitored as its high-spin spectral (type I) complex, but had little effect on the CYP3A4 binding of either α-naphthoflavone or testosterone [80]Given that GSH is routinely included in CYP3A4 reconstitution assays at relatively high concentrations [81], it is to be underscored, that the CYP3A4-hemeiron-GSH interactions detected at 1-10 mM concentrations, while decreasing 1-PB and 7-BFC homotropic cooperativity, failed to competitively inhibit these substrates, and if at all increased their binding affinity (1-PB) and/or their activity (7-BFC) [80] Such failure of GSH (unlike that of the organic lipophilic thiols and sulfide agents discussed above) to effectively compete out other substrates in functionally reconstituted CYP3A4 systems, may be due to its relatively lower lipophilicity and consequently lower affinity for the lipophilic CYP3A4 active site, coupled with the expected dissociation that ensues from the P450 heme iron upon CPR-mediated reduction and subsequent competition with O 2 binding…”

Current Approaches for Investigating and Predicting Cytochrome P450 3A4-Ligand Interactions

“…Analysis of the substrate- and probe-dependent fluorescence changes and H 2 O 2 -induced heme destruction kinetics provided evidence for a distinct low-affinity ANF binding site, association to which is not accompanied by a heme spin shift. BADAN-labeled CYP3A4 was also used for investigating the effects of reduced glutathione (GSH) on BFC and BQ oxidation [52]. GSH eliminates homotropic cooperativity of BFC and BQ but amplifies the activating effect of ANF on BFC oxidation, and is suggested to have two binding modes, one of which is direct coordination to the heme iron via the SH-group.…”

Microsomal monooxygenase as a multienzyme system: the role of P450-P450 interactions

“…Therefore, modulation of homo- and hetero-oligomerization of cytochromes P450 by peripherally-bound allosteric effectors may be considered as a possible allosteric regulatory mechanism that underlies heterotropic cooperativity [122]. It was hypothesized that the physiological regulation of MMO through this mechanism may involve such endogenous effectors as reduced glutathione (GSH) [122, 129]. …”