No abstract

No abstract

To explore the basis of apparent conformational heterogeneity of cytochrome P450 3A4 (CYP3A4), the kinetics of dithionite-dependent reduction was studied in solution, in proteoliposomes, and in Nanodiscs. In CYP3A4 oligomers in solution the kinetics obeys a three-exponential equation with similar amplitudes of each of the phases. Addition of substrate (bromocriptine) displaces the phase distribution toward the slow phase at the expense of the fast one, while the middle phase remains unaffected. The fraction reduced in the fast phase, either with or without substrate, is represented by the low-spin heme protein only, while the slow-reducible fraction is enriched in the high-spin CYP3A4. Upon monomerization by 0.15% Emulgen-913, or by incorporation into Nanodiscs or into large proteoliposomes with a high lipid-to-protein (L/P) ratio (726:1 mol/mol), the kinetics observed in the absence of substrate becomes very rapid and virtually monoexponential. In Nanodiscs and in lipid-rich liposomes bromocriptine decreases the rate of reduction via appearance of the second (slow) phase, the amplitude of which reaches 100% at saturating bromocriptine. In contrast, in P450-rich liposomes (L/P = 112 mol/mol), where the surface molar density of the enzyme is comparable to that observed in liver microsomes, CYP3A4 behaves similarly to that observed in solution. These results suggest that in CYP3A4 oligomers in solution and in the membrane the enzyme is distributed between two persistent conformers with different accessibility of the heme for the reductant (SO*-(2) anion monomer). One of the apparent conformers exists in a substrate-dependent equilibrium between two states with different rate constants of reduction by dithionite, while the second conformer shows no response to substrate binding.

No abstract

According to the initial hypothesis on the mechanisms of cooperativity in drug-metabolizing cytochromes P450, a loose fit of a single substrate molecule in the P450 active site results in a requirement for the binding of multiple ligand molecules for efficient catalysis. Although simultaneous occupancy of the active site by multiple ligands is now well established, there is increasing evidence that the mechanistic basis of cooperativity also involves an important ligandinduced conformational transition. Moreover, recent studies demonstrate that the conformational heterogeneity of the enzyme is stabilized by ligand-dependent interactions of several P450 molecules. Application of the concept of an oligomeric allosteric enzyme to microsomal cytochromes P450 in combination with a general paradigm of multiple ligand occupancy of the active site provides an excellent explanation for complex manifestations of the atypical kinetic behavior of the enzyme.

Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into α-helix A‥ The site of modification was identified as Cys-64 with the help of CYP3A4 (C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled with 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN), 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromobimane (mBBr) were used to study the interactions with bromocriptine (BCT), 1-pyrenebutanol (1-PB), testosterone (TST), and α-naphthoflavone (ANF). Of these substrates only ANF has a specific effect, causing a considerable decrease in fluorescence intensity of BADAN and CPM and increasing the fluorescence of mBBr. This ANF-binding event in the case of BADAN-modified enzyme is characterized by an S 50 of 18.2 ± 0.7, compared with the value of 2.2 ± 0.3 for the ANF-induced spin transition, thus revealing an additional low affinity binding site. Studies of the effect of TST, 1-PB, and BCT on the interactions of ANF monitored by changes in fluorescence of CYP3A4(C58,C64)-BADAN or by the ANF-induced spin transition revealed no competition by these substrates. Investigation of the kinetics of fluorescence increase upon H 2 O 2 -dependent heme depletion suggests that labeled CYP3A4(C58,C64) is represented by two conformers, one of which has the fluorescence of the BADAN and CPM labels completely quenched, presumably by photoinduced electron transfer from the neighboring Trp-72 and/or Tyr-68 residues. The binding of ANF to the newly discovered binding site appears to affect the interactions of the label with the above residue(s), thus modulating the fraction of the fluorescent conformer. KeywordsCytochrome P450 3A4; α-naphthoflavone; cooperativity; substrate binding site; BADAN; conformational heterogeneity A number of recent studies on the mechanisms of function and regulation of microsomal monooxygenase systems have focused on the importance of homo-and heterotropic cooperativity exhibited by various mammalian drug-metabolizing cytochromes P450 (1-8). Cooperative behavior has been reported for such enzymes as P450 1A2 (9), 2C9 (10-12), 2C5 (13), 2B6 (14), and 2B4 (15). However, the most prominent cases of P450 cooperativity are found in cytochrome P450 3A4 (CYP3A4), the principal drug-metabolizing enzyme in humans † This research was supported by NIH grant GM54995, Center grant ES06676, and research grant H-1458 from the Robert A. Welch NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 October 28. Published in final edited form as:Biochemistry. 2007 January 9; 46(1): 106-119. doi:10.1021/bi061944p. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (16). In addition to the homotropic cooperativity observed with a wide variety of substrates (1,4,(16)(17)(18), CYP3A4 also provides important examples of heterotropic activation (1...

SYNOPSIS We investigated the relationship between oligomerization of cytochrome P450 3A4 (CYP3A4) and its response to α-naphthoflavone (ANF), a prototypical heterotropic activator. Addition of ANF resulted in over a two-fold increase in the rate of CYP3A4-dependent debenzylation of 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC) in human liver microsomes (HLM) but failed to produce activation in BD Supersomes™ or Baculosomes® containing recombinant CYP3A4 and NADPH-cytochrome P450 reductase (CPR). However, incorporation of purified CYP3A4 into Supersomes containing only recombinant CPR reproduced the behavior observed with HLM. The activation in this system was dependent on the surface density of the enzyme. While no activation was detectable at a lipid:P450 (L/P) ratio ≥ 750, it reached 225% at an L/P ratio of 140. To explore the relationship between this effect and CYP3A4 oligomerization we probed P450-P450 interactions with a new technique based on luminescence resonance energy transfer (LRET). The amplitude of LRET in mixed oligomers of the heme protein labeled with donor and acceptor fluorophores exhibited a sigmoidal dependence on the surface density of CYP3A4 in Supersomes. Addition of ANF eliminated this sigmoidal character and increased the degree of oligomerization at low enzyme concentrations. Therefore, the mechanisms of CYP3A4 allostery with ANF involve effector-dependent modulation of P450-P450 interactions.

To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continous variation (Job's titration) experiments allowed us to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the Type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 ± 0.3 (S 50 = 8.0 ± 1.1 µM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting K D values of 0.31 ± 0.08 µM and 6.7 ± 1.9 µM, respectively. The binding of all three substrates monitored by FRET in titration-by-dilution experiments at an enzyme:substrate ratio of 1 reveals a simple bimolecular interaction with K D values of 0.16 ± 0.09, 4.8 ± 1.4, and 0.18 ± 0.09 µM for 1-PB, PMA, and bromocriptine respectively. Correspondingly, the Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of K D obtained in our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site.Keywords substrate binding; cooperativity; conformers; spin equilibrium Recent studies of function and regulation of cytochromes P450 reveal increasing attention to the mechanisms and pharmacological significance of homo-and heterotropic cooperativity observed in various mammalian P450 species (1-8). The most prominent examples of these phenomena are observed with cytochrome P450 3A4 (CYP3A4), the most abundant P450 in human liver. CYP3A4 is responsible for the metabolism of a broad range of drug substrates, and studies of cooperativity of this enzyme are of vital importance to our efforts to elucidate the mechanisms of drug metabolism in humans (9). However, despite extensive studies, a general mechanism of cooperativity remains obscure. † This research was supported by NIH grants GM54995 (JRH), and Center grant ES06676 (JRH). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe prevailing hypothesis is that cytochromes P450 exhibiting cooperativity accommodate multiple substrate molecules in one large binding pocket (10)(11)(12). A loose fit of a single substrate molecule requires the binding of a second ligand for efficient binding and/or catalysis (11-13). However, the possibility that P450 cooperativity reflects a true case of a...