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Originally published In Press as doi:10.1074/jbc.M511375200 on February 8, 2006

J. Biol. Chem., Vol. 281, Issue 14, 9127-9136, April 7, 2006
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Kinetics and Thermodynamics of Ligand Binding by Cytochrome P450 3A4*Formula

Emre M. Isin and F. Peter Guengerich1

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Received for publication, October 19, 2005 , and in revised form, January 3, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytochrome P450 (P450) 3A4, the major catalyst involved in human drug oxidation, displays substrate- and reaction-dependent homotropic and heterotropic cooperative behavior. Although several models have been proposed, these mainly rely on steady-state kinetics and do not provide information on the contribution of the individual steps of P450 catalytic cycle to the observed cooperativity. In this work, we focused on the kinetics of substrate binding, and the fluorescent properties of bromocriptine and {alpha}-naphthoflavone allowed analysis of an initial ligand-P450 3A4 interaction that does not cause a perturbation of the heme spectrum. The binding stoichiometry for bromocriptine was determined to be unity using isothermal titration calorimetry and equilibrium dialysis methods, suggesting that the ligand bound to the peripheral site during the initial encounter dissociates subsequently. A three-step substrate binding model is proposed, based on absorbance and fluorescence stopped-flow kinetic data and equilibrium binding data obtained with bromocriptine, and evaluated using kinetic modeling. The results are consistent with the substrate molecule binding at a site peripheral to the active site and subsequently moving toward the active site to bind to the heme and resulting in a low to high spin iron shift. The last step is attributed to a conformational change in the enzyme active site. The later steps of binding were shown to have rate constants comparable with the subsequent steps of the catalytic cycle. The P450 3A4 binding process is more complex than a two-state system, and the overlap of rates of some of the events with subsequent steps is proposed to underlie the observed cooperativity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytochrome P450 (P450)2 enzymes are found throughout nature, from bacteria to humans. These enzymes generally catalyze mixed function oxidation reactions that have similar chemistry or else utilize parts of the general catalytic mechanism for reductions and rearrangements (2, 3). The wide diversity of substrates of these enzymes and the basis of catalytic selectivity is a topic of considerable interest in the context of both basic biochemistry and practical applications (4).

P450 3A4 is one of the most widely studied of the 57 human P450s (5), mainly due to its role in the metabolism of more than one-half of the drugs on the market as well as various endogenous and exogenous molecules (6, 7). In addition, P450 3A4 is the major P450 expressed in liver (8) and in the intestine (9). Recently solved P450 3A4 crystal structures (10–12) demonstrate the presence of a large active site, consistent with the broad range of substrates that P450 3A4 can accommodate (7, 13), including (in order of increasing size) acetaminophen (14) (Mr 151), testosterone (15) (Mr 288), bromocriptine (16) (Mr 655), and cyclosporin (17) (Mr 1201). Despite its seemingly flexible substrate selectivity, P450 3A4 displays a high degree of regio- and stereoselectivity in many substrate oxidations (18, 19).

One of the important features of P450 3A4 is its cooperative behavior, manifested in unusual substrate oxidation kinetics. One of the earlier examples is the stimulation of the 8,9-epoxidation of aflatoxin B1 by {alpha}-naphthoflavone (20–22), which itself is a P450 3A4 substrate. Following this finding, many examples of cooperativity have been reported not only for P450 3A4 but also for human P450s 2C9 (23), 1A2 (24, 25), and 2B6 (26), providing support that cooperativity is a common feature of multiple P450s (27) and that the effects are substrate- and reaction-dependent (28). Testosterone, 17beta-estradiol, aflatoxin B1, and amitriptyline were shown to display homotropic cooperativity (29) (i.e. increasing substrate concentration stimulates oxidation, resulting in sigmoidal velocity versus substrate concentration curves) (30). Various flavonoids have been shown to act as effectors to stimulate the oxidation of some substrates, resulting in heterotropic cooperativity (30), and to inhibit the oxidation of others (31). Thus, the interaction of P450 3A4 with ligands is quite complex and can have a significant effect on the observed in vitro oxidation kinetics.

Examples of in vivo cooperativity in animal models have been developed (32), and an understanding of the underlying mechanisms of cooperative behavior is important in prediction of drug-drug interactions in practical settings (33). Several groups have proposed models to explain the observed cooperativity (34). A rather general consensus is that multiple ligands may interact with P450 3A4 simultaneously, although (i) direct physical evidence is very limited (35) and (ii) the number of substrate/effector molecules bound and binding sites has not been established. One model, based on steady-state kinetics, has simultaneous occupancy of the P450 3A4 active site with two substrates, which may or may not be identical molecules and which are bound to different domains of the active site (36–38). Alternatively, a three-site model with a distinct effector binding site has been proposed using a similar steady-state kinetic modeling approach (39). Site-directed mutagenesis studies have led to the proposal of a two-substrate/one-effector domain model in which the effector is located in close proximity of the active site (40, 41). However, all of these models are based on steady-state turnover kinetic data and do not provide detailed information on the contributions of the individual steps of the catalytic cycle to the observed cooperativity. We have previously focused on the substrate binding step of the P450 catalytic cycle in an attempt to understand better the mechanisms involved in cooperativity (42). Recent work from two other groups has also been directed toward substrate binding to P450 3A4 using spectral and EPR approaches (43, 44).

In this work, we have expanded our investigations of ligand binding to P450 3A4 (29, 42) using transient state kinetics and equilibrium methods, including quantitative dialysis and ITC. Ligand fluorescence was used as a direct approach to monitor ligand-P450 3A4 interactions, in addition to UV-visible observations of heme perturbations. Our studies on the interactions of various molecules with P450 3A4 lead us to propose a three-step binding model in which the first step does not perturb the heme spectrum. We have also examined the relevance of the individual steps of ligand binding to reduction of ferric to ferrous P450 subsequent event in the catalytic cycle of P450 3A4. These studies demonstrate that binding of ligands to P450 3A4 is a complex multistep process and that investigation of ligand binding interactions solely with heme spin state changes can be misleading.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—{alpha}-Naphthoflavone, midazolam, flavone, and bromocriptine were purchased from Sigma. Testosterone was obtained from Steraloids (Newport, RI). All other reagents and solvents were obtained from general commercial suppliers. All chemicals were used without further purification.

Spectroscopy—Absorbance spectra were recorded using either an Aminco DW2a/OLIS or a Cary 14/OLIS spectrophotometer (On-Line Instrument Systems, Bogart, GA). Fluorescence measurements were made using a DM45/OLIS spectrofluorimeter. Stopped-flow experiments were carried out using an OLIS RSM-1000 instrument. A 4 x 20-mm cell was used for absorbance measurements, and a 4 x 4-mm cell was used for fluorescence measurements.

Enzymes—Recombinant P450 3A4 with a C-terminal His5 tag (45) was expressed in Escherichia coli and purified as described previously (42). E. coli recombinant rat NADPH-P450 reductase (46) and human liver cytochrome b5 (47) were prepared as described elsewhere. Protocatechuate dioxygenase was a gift from D. P. Ballou (University of Michigan, Ann Arbor, MI).

Reconstitution System—For the reduction kinetics experiments and for some of the testosterone binding experiments, P450 3A4 was reconstituted freshly before the experiment by mixing the components in the following order: P450 3A4 (0.5 or 2 µM), NADPH-P450 reductase (1 or 4 µM), cytochrome b5 (0.5 or 2 µM), sodium cholate (0.5 mM), and phospholipid mixture (40 µg/ml, prepared as described previously (48)). The components were kept at room temperature for 20 min and mixed periodically by gentle shaking. After 20 min, potassium HEPES buffer (50 mM, pH 7.4), glutathione (3 mM), and MgCl2 (30 mM) were added, and the reconstituted system was kept on ice until use.

Spectral Binding Titrations—Binding affinities of ligands to P450 3A4 were determined (at 23 °C) by titrating 1 µM enzyme with the ligand, in a total volume of 1.0 ml of 100 mM potassium phosphate buffer (pH 7.4). Final CH3OH concentrations were ≤2% (v/v). The reference cuvette, containing an equal concentration of enzyme in buffer, was titrated with an equal volume of the vehicle solvent. UV-visible spectra (350–500 nm) were recorded after each addition, and the absorbance differences (at the wavelength maximum and minimum) were plotted against the added ligand concentrations. Spectral dissociation constants (Ks) were estimated using GraphPad Prism software (GraphPad Software, San Diego, CA) or DynaFit (49) simulation software (Biokin, Pullman, WA). Unless the estimated Ks was within 5-fold of the P450 concentration, a nonlinear regression analysis was applied using the hyperbolic equation {Delta}A = Bmax[L]/(Ks + [L]), where A is the absorbance difference, Bmax is the maximum absorbance difference extrapolated to infinite ligand concentration, and [L] is the ligand concentration. For the high affinity ligand bromocriptine, a quadratic equation was used to correct for the bound enzyme concentration: {Delta}A = A0 + (Bmax/2[E])((Ks + [E] + [L]) – ((Ks + [E] + [L])2 – 4[E][L])1/2), with E being the total enzyme concentration and A0 being a coefficient in each analysis and not relevant. Alternatively, DynaFit simulation software was used with a one-step enzyme-ligand binding model, yielding identical results obtained from the hyperbolic or quadratic nonlinear regression analyses. With flavone, the best fit was obtained with the Hill equation Formula, where n is a measure of cooperativity.

Binding Kinetics of Ligands to P450 3A4—In the stopped-flow experiments, either the changes in heme spectra were monitored as a function of time (in the absorbance mode), or the fluorescence quenching of ligands was monitored (in the fluorescence mode). One of the drive syringes contained purified P450 3A4, diluted to 2 or 4 µM in 100 mM potassium phosphate buffer (pH 7.4). The second drive syringe contained the ligand solution (bromocriptine, dissolved in 0.10 M HCl, was diluted in buffer to the desired concentration, and all other ligands were dissolved in CH3OH with ≤2% (v/v) final CH3OH concentration). All stopped-flow measurements were carried out at 23 °C. Immediately after mixing equal amounts (150 µl) of reagent from both syringes, UV-visible spectra (350–500 nm) were collected in the rapid scanning mode with a 16 x 1-mm scan disk, which is a component of the instrument and is used to acquire spectra rapidly. Depending on the data collection time, between 10 and 1000 scans/s were acquired; generally, averages of four experiments were used in the subsequent data analyses. Time-resolved spectra were collected with at least five ligand concentrations for each ligand. Kinetic traces were extracted from the acquired spectra, utilizing either {Delta}A390 or {Delta}A390 – A418 (or {Delta}A390 – A420) and were analyzed using the manufacturer's software (OLIS), GraphPad Prism, or DynaFit.

Binding kinetics of testosterone and bromocriptine to P450 3A4 were also measured using the reconstituted system. In these experiments, one of the drive syringes contained P450 3A4 (2 µM), NADPH-P450 reductase (4 µM), cytochrome b5 (2 µM), and other components of the reconstitution system. The second drive syringe contained the testosterone or the bromocriptine solution.

In the fluorescence experiments, after mixing the contents of the drive syringes, emission spectra were collected as a function of time with a midplane photomultiplier tube and using the 16 x 1-mm scan disk. The excitation wavelength was 325 nm for bromocriptine and {alpha}-naphthoflavone. In general, an excitation monochromator slit of 1.24 mm was used, corresponding to an 8-nm bandpass. Alternatively, data were collected using a >385-nm long pass filter in the single wavelength mode, and the kinetic traces were analyzed using the OLIS software or GraphPad Prism. Generally, averages of four experiments were used in the subsequent data analyses, and S.E. values indicate the goodness of the fit to the average of the multiple experiments.

Anaerobic Experiments—The basic set-up (50) and recent modifications (51) have been described elsewhere. Anaerobic conditions were achieved by connecting the glass tonometers to a gas train via a manifold and alternating between vacuum and argon for 10 cycles. In the reduction experiments, an additional three vacuum-CO cycles were carried out prior to loading the drive syringes. An O2 scrubbing system was included in the glass tonometers consisting of protocatechuate dioxygenase (0.7 µM) and protocatechuate (80 µM, added into the solution after 5 vacuum/argon cycles, through a side arm) (52). The drive syringes and lines of the stopped-flow apparatus were depleted of O2 using an overnight procedure described in detail previously (53).

Reduction Kinetics—The reduction rate of ferric (Fe3+) to ferrous (Fe2+) P450 was measured at 23 °C with the increase in absorbance at 450 nm or the decrease at 390 nm. Glass tonometers (under a positive CO atmosphere and containing the O2 scrubbing system; see above) were used to fill the drive syringes of the stopped-flow instrument, with minimum exposure to the atmosphere. One of the syringes contained an anaerobic solution of P450 3A4 (0.5 µM, reconstituted as described above), and the second syringe contained 0.4 mM NADPH. In some reduction experiments, bromocriptine (10 µM) was also included either in the syringe containing the enzyme or the one containing NADPH. After mixing the components of the two syringes, UV-visible spectra (355–580 nm) were collected, and kinetic traces were extracted at the wavelengths of interest and analyzed further using GraphPad Prism. As in the case of binding kinetics experiments, between 10 and 1000 scans/s were acquired, depending on the data collection time, and generally averages of four experiments were used in the subsequent data analyses.

Equilibrium Dialysis—The stoichiometry of bromocriptine binding to P450 3A4 was examined using five-cavity equilibrium dialysis cells (Bel-Art Products, Pequannock, NJ). A dialysis membrane with a 12–14-kDa cut-off was placed between the two halves of the dialysis cell. The solutions (900 µl) were loaded into the cavities using a 1-ml syringe. Both sides contained an equal amount of bromocriptine solution (0–5 µM in 100 mM potassium phosphate buffer, pH 7.4, diluted from a 300 µM bromocriptine stock solution in 0.10 M HCl). In addition to bromocriptine, one side also contained P450 3A4 (1.0 µM). The solutions were equilibrated for 24 h at room temperature with mechanical rocking. After equilibration, 800-µl aliquots were removed from the cavities, and 100 µl of 25% HClO4 was added. The solutions were mixed using a vortex device and centrifuged (2 x 103x g, 10 min), and 750-µl aliquots were removed. The aliquots were diluted to 2.0 ml with H2O, and fluorescence spectra (350–550 nm) were recorded using mirror-coated fluorimeter cuvettes (Starna Cells, Atascadero, CA). The excitation wavelength was 325 nm, and emission at 420 nm was used to quantify the amount of bromocriptine.

ITC—ITC titrations were carried out at 25 °C using a VP-ITC instrument (MicroCal, Northampton, MA). Prior to the titration, P450 3A4 was dialyzed twice against 100 volumes of 100 mM potassium phosphate buffer (pH 7.4) at 4 °C for 4 h. Following the dialysis, the P450 concentration was determined spectrally using the method of Omura and Sato (54). The reference cell of the ITC instrument was filled with dialysis buffer. In a typical experiment, the bromocriptine solution (5 µM in dialysis buffer, diluted from 300 µM bromocriptine stock solution in 0.10 M HCl) in the ITC cell was titrated with P450 3A4 (35 µM) loaded into the ITC syringe. The first injection (2 µl, omitted from analysis) was followed by 25 injections of 10 µl, with 8-min intervals between injections. The cell contents were stirred at 450 rpm to provide immediate mixing. The thermal power (heat per unit time) required to keep the cell temperature constant was monitored with time. The peaks observed in the power versus time plots (thermograms, not shown) were integrated using the ORIGIN software (MicroCal). The heats of dilution were obtained from the saturating part of the thermograms and subtracted from each integrated peak. The total heat change was plotted versus the concentration of P450 added to give a titration curve (binding isotherm), expressed in units of kJ mol–1.

Kinetic Modeling of Data—Kinetic binding data were fit to various proposed models using DynaFit software (49). Rate constants were estimated by globally fitting the kinetic data at six different ligand concentrations to the proposed models. The initial ligand concentrations were allowed to float within 10% of the starting value in the system, and in most cases the concentration adjustment was less than 5%. Sample scripts are included in the supplemental data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Spectral Equilibrium Binding Titrations—Affinities of various ligands with P450 3A4 were first estimated spectrophotometrically by monitoring the heme spectral changes upon the addition of ligands. Ks values were obtained from the titration curves, as described under "Experimental Procedures." Ks values estimated previously in this laboratory and the Ks values determined for the new ligands included in this study are summarized in Table 1.


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  		TABLE 1
Binding rates and affinities of ligands to P450 3A4

 
The P450 3A4 substrates testosterone, midazolam, flavone, {alpha}-naphthoflavone, and bromocriptine all produced a Type I shift in the heme Soret band, reflecting the displacement of H2O as the sixth ligand and resulting in a low to high spin change in the P450 iron (55). Bromocriptine, the most bulky of the substrates studied, had the highest apparent affinity (Ks = 0.4 µM), consistent with a previously reported dissociation constant (16). The titration plot obtained for flavone binding to P450 3A4 displayed apparent sigmoidicity (supplementary data). A fit to the Hill equation resulted in a Ks (S50) value of 27 µM and a coefficient (n) of 1.9, suggesting a high degree of binding cooperativity. A structurally related molecule, {alpha}-naphthoflavone, has also been shown to display cooperative binding to P450 3A4 with a Ks (S50) of 5.7 µM and an n value of 1.7 (42).

Kinetics of Ligand Binding—Binding kinetics of ligands to P450 3A4 were investigated using absorbance measurements at different ligand concentrations. The spectra, acquired as a function of time, displayed Type I patterns with an increase in absorbance at 390 nm and a decrease in absorbance at 418–420 nm, as expected (Fig. 1).


Figure 1
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  		FIGURE 1.
Binding of testosterone to P450 3A4. A, spectral changes observed with time after mixing 4 µM P450 3A4 with 200 µM testosterone in the stopped-flow apparatus. The traces shown were collected at 0, 20, 40, 60, 80, 120, 140, and 160 ms. B, kinetic traces extracted at 390 and 420 nm from spectra acquired over time (A). C, rate of {Delta}A390 for testosterone binding fit to a biexponential plot with 37 and 3.8 s–1. Analysis of residuals for the biexponential fit is shown at the top.

 
The kinetic traces extracted at 390 nm for substrates (Fig. 1B) were used to estimate the rates of binding. For all examined ligands, single exponential fits proved unsatisfactory and suggested kinetics that are at least biphasic. At increasing ligand concentrations, the multiphasic nature of the kinetics became more apparent. The kinetic data were fit to a biexponential equation (Fig. 1C), and the observed rates of binding in the two phases (ka and kb) at saturating ligand concentrations are summarized in Table 1. Biexponential fits were satisfactory for most of the ligands, as judged by the residual analyses (top of Fig. 1C). The fitted rates of the first observed binding component were plotted versus the ligand concentration in each case to estimate the apparent second-order rate constants (Table 1). We did not attempt to use these results to estimate k–1, the off rate. The calculated rates are less than expected for diffusion-limited encounters of substrates with ligands (56) and the rates seen in other P450 systems (53, 57, 58) and suggest complexity in the system.

With flavone, an unusual kinetic behavior was observed with a rapid increase in absorbance at 390 nm and a decrease at 418 nm (Type I) shift, followed by a second slower phase in which a reverse Type I shift was observed (with a decrease in absorbance at 390 nm accompanied by an increase at 418 nm) (Fig. 2). The rates for the two phases at saturating flavone concentration (100 µM) were estimated to be 46 and 0.8 s–1, respectively, using a single exponential fit for each individual phase.


Figure 2
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  		FIGURE 2.
Binding kinetics of flavone to P450 3A4. A, spectral changes observed with time after mixing 4 µM P450 3A4 with 200 µM flavone in the stopped-flow apparatus. The traces shown were collected at 0 (base line), 16 (solid line), 96 (thick solid line), 1200 (dotted line), and 3200 (thick dotted line) ms. B, kinetic traces extracted at 390 and 418 nm from spectra acquired as a function of time after mixing (from A).

 
The binding kinetics of testosterone and bromocriptine in a reconstituted P450 3A4 system (with NADPH-P450 reductase and cytochrome b5) were also investigated, The rates of binding at 100 µM testosterone were found to be ~3-fold faster compared with P450 3A4 alone (ka = 110 s–1, kb = 13 s–1). The rates estimated for bromocriptine binding at 5 µM were 16 and 1.1 s–1. Although binding events seem to take place faster in the reconstituted system, the kinetic behavior was qualitatively identical to the system containing only the P450.

Fluorescence Quenching of Ligands upon Interaction with P450 3A4—Of the investigated ligands, bromocriptine and {alpha}-naphthoflavone are fluorescent, and the examination of ligand-P450 3A4 interactions was possible using kinetic analysis with this spectral method. Approximately 7% of the total bromocriptine fluorescence was quenched rapidly, at a rate of 20 s–1, followed by a slower phase in which another 6% of the initial fluorescence was quenched at a rate of 0.6 s–1 (Fig. 3A). The bimolecular rate constants for the first step (k1 = 4.0 x 106 M–1 s–1, k–1 = 4 s–1) were estimated from a plot of rates versus bromocriptine concentrations. This rate is considerably faster than measured with absorbance methods (Table 1). When a solution of bromocriptine premixed with P450 3A4 was mixed with the ligand indinavir (20 µM) (42, 59) in the stopped-flow apparatus, the fluorescence emission of bromocriptine was partially restored (Fig. 3B). In an analogous experiment, the addition of testosterone (100 µM) also increased the bromocriptine fluorescence (results not shown). These results demonstrate the reversibility of the system and argue that the decrease in fluorescence is not an artifact.


Figure 3
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  		FIGURE 3.
Bromocriptine fluorescence quenching upon binding to P450 3A4. A, decay in bromocriptine fluorescence emission (>385 nm) upon mixing 2 µM P450 3A4 with 10 µM bromocriptine in the stopped-flow apparatus. The kinetic trace is fit to a biexponential plot with rates of 20 and 0.6 s–1. The fast phase (first 200 ms) of fluorescence decay is shown in the inset. Analysis of residuals for the biexponential fit is shown at the top. B, recovery of bromocriptine fluorescence (emission at >385 nm) upon mixing of 20 µM indinavir with a preformed mixture of 2 µM P450 3A4 and 10 µM bromocriptine, demonstrating the reversibility of the fluorescence change.

 
The fluorescence of {alpha}-naphthoflavone was quenched rapidly upon mixing with P450 3A4. When the fluorescence decay of {alpha}-naphthoflavone was monitored over a period of 1 s, biphasic kinetic behavior was also observed. In the fast phase, 14% of the initial fluorescence was quenched (at a rate of 86 s–1), and another 9% of the initial fluorescence was quenched in the slow phase (at a rate of 4.5 s–1) (supplemental data). Rates were estimated at varying concentrations of ligand and fit to yield a second-order rate of 2.6 x 107 M–1 s–1.

Determination of Binding Stoichiometry of Bromocriptine to P450 3A4—Because the initial rapid enzyme encounter is observable only by fluorescence but not with absorbance (hence "silent"), bromocriptine does not appear to reach a position close enough to the P450 3A4 active site to perturb the heme spectra within this time frame. Therefore, it is possible that a second molecule of bromocriptine may be moving toward the heme to cause the observed Type I change, whereas the first molecule stays bound to an "outer" site (where the initial interaction occurs). In order to investigate further the possibility of a second molecule bound to P450 3A4, particularly in light of suggestions in the literature (35–42), equilibrium dialysis and ITC approaches were used to determine the binding stoichiometry.


Figure 4
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  		FIGURE 4.
Stoichiometry of bromocriptine binding to P450 3A4. A, equilibrium dialysis. The contents of the two compartments of the microdialyzer system separated by dialysis membrane were equilibrated for 24 h. Both compartments contained equal concentrations of bromocriptine initially. One compartment contained 1.0 µM P450 3A4 in addition to bromocriptine. The concentration of bound bromocriptine is plotted against the free bromocriptine concentration. B, ITC. The ITC cell contained 5 µM bromocriptine, and the titration syringe contained 36 µM P450 3A4. The total {Delta}H (kJ/mol of P450 added) was plotted versus the concentration of P450 3A4 added.

 
In the equilibrium dialysis experiments, more bromocriptine was present in the compartment containing the enzyme than the compartment containing only bromocriptine (although the total recovery of bromocriptine was less than quantitative). A titration curve was constructed by plotting the bound versus free bromocriptine concentration after equilibration (Fig. 4A). A plateau was reached at a concentration of 1 µM for bound bromocriptine, suggesting 1:1 stoichiometry with P450 3A4.

ITC was used to determine the binding stoichiometry in an independent approach, based on the changes in the heat of binding. Because of the low solubility of the ligands in the dialysis buffer, bromocriptine was placed in the ITC cell rather than in the titration syringe. Thus, a low concentration (5 µM) of bromocriptine can be titrated to saturation with a high concentration (36 µM) of P450 3A4. The binding event was exothermic, as frequently observed for other enzyme-ligand binding interactions (60–62). When the total heat change was plotted versus the increasing P450 3A4 concentration in the cell (Fig. 4B), a plateau was observed at ~6 µM P450, corresponding approximately to a 1:1 stoichiometry between bromocriptine and P450 3A4 and consistent with the results of the equilibrium dialysis experiment.

Relevance of Binding Rates to the Substrate Stimulation of Reduction of Ferric to Ferrous P450—Reduction of ferric to ferrous P450 by NADPH-P450 reductase is the step following the binding of substrate to P450 3A4 and has been shown previously to be highly stimulated in the presence of substrates (48, 63). Because the rate obtained for the last step of binding of bromocriptine is slower than previously reported rates of reduction (e.g. 12 s–1 at 37 °C in the presence of 100 µM testosterone (63)), it is possible that the completion of the slow binding step may be a requirement for the stimulation of the reduction step. Alternatively, the first two steps (or only the first step) of binding may be sufficient to facilitate the reduction step.

A series of stopped-flow reduction experiments were carried out at 23 °C under anaerobic conditions to examine the effect of individual steps of binding on the rate of reduction (Fig. 5). In the absence of substrate (results not shown), reduction proceeded very slowly at a rate of ~0.15 s–1, as expected (48, 63). When a premixed solution of bromocriptine (10 µM) plus P450 3A4 was anaerobically reduced with NADPH, a rate of 3.9 s–1 was measured (Fig. 5A). In a third experiment, a solution of P450 3A4 was mixed with a solution of NADPH containing 10 µM bromocriptine under anaerobic conditions. The rate of reduction in this experiment was ~0.2 s–1 (Fig. 5B, single-exponential fit is not shown), similar to the rate of reduction in the absence of substrate, suggesting that the last binding step for bromocriptine has to be completed for the stimulation of the reduction.


Figure 5
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  		FIGURE 5.
Reduction kinetics of ferric P450 3A4 in the presence of bromocriptine. The reduction of P450 3A4 was monitored at 450 nm. Experiments were done anaerobically under a CO atmosphere by mixing reconstituted P450 3A4 with NADPH in the stopped-flow apparatus. A, one syringe contained 0.5 µM P450 3A4, reconstituted as described under "Experimental Procedures," plus 10 µM bromocriptine. The second syringe contained an equal volume of 0.4 mM NADPH. Both syringes contained the additional reaction components (see "Experimental Procedures") to facilitate the removal of O2 from the system. The rate of {Delta}A450 fit to a single exponential with 3.9 s–1. Analysis of residuals for the single exponential fit is shown at the top. B, one syringe contained 0.5µM P450 3A4 reconstituted as described under "Experimental Procedures." The second syringe contained an equal volume of 0.4 mM NADPH and 10 µM bromocriptine. As in A, both syringes contained additional reaction components (see "Experimental Procedures") to facilitate the removal of O2 from the system. (The short lag at the start of each reaction is attributed to the decrease in flavin absorbance of the NADPH-P450 reductase upon mixing).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The focus of this study was the kinetics of ligand binding with P450 3A4. We monitored the changes in P450 3A4 heme spectra during ligand binding using time-resolved stopped-flow absorbance. In a simple one-step binding process (E + S {rightleftarrows} ES), ligand binding kinetic data are expected to fit a single exponential, as shown for the interaction of P450 101A1 with camphor (57) and coumarin with P450 2A6 (53). However, biphasic kinetics were clearly observed for binding of all studied substrates to P450 3A4 (Fig. 2C and Table 1), as judged by {Delta}A390 measurements, suggesting a multistep binding process (64). Also, the rates obtained using a biexponential fit were significantly slower than expected for diffusion-controlled ligand-enzyme encounters (i.e. ~107 M–1 s–1) (56). These results led us to consider the presence of an initial step that does not cause the perturbation of heme spectra and is therefore not detectable by monitoring absorbance changes. An alternative approach to following ligand-P450 interactions involves monitoring changes in fluorescence emission of the ligand, without relying on heme spectral changes. Fluorescence stopped-flow experiments revealed that bromocriptine fluorescence was rapidly quenched upon mixing with P450 3A4, with biphasic kinetics (Fig. 3A). The rate obtained for the first phase was significantly faster than the rate observed via absorbance measurements at the same concentration, providing direct evidence for an initial encounter between bromocriptine and P450 3A4 that does not cause any changes in heme spectra. The second fluorescent step has a rate (0.6 s–1), quite similar to the first phase detected in the absorbance measurements (1 s–1 at 5 µM bromocriptine). Stopped-flow fluorescence experiments carried out with {alpha}-naphthoflavone yielded similar results (supplemental data).


Figure 6
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  		FIGURE 6.
Proposed model of substrate binding to P450 3A4. In the first step, ligand binds further away from the heme (1). Following this initial rapid encounter, either the ligand molecule bound at the peripheral site translocates toward the heme (2a), or a second ligand molecule enters the active site and interacts with the heme (2b). The third step is attributed to a conformational change in both pathways (3).

 
These results are consistent with a three-step model (Fig. 6), in which the ligand molecule binds distantly from the heme, possibly at a site peripheral to the recognized substrate cavity. This initial encounter is rapid and not detectable by absorbance spectroscopy. Although we do not have sufficient information to determine the location of this binding site, it is possible that the initial encounter may be occurring where a progesterone molecule was found to be bound in one of the P450 3A4 crystal structures (11). Following the initial encounter, the ligand molecule translocates toward the heme, causing a low to high spin iron transition, which is the faster phase detected by stopped-flow absorbance and also the slower phase of fluorescence quenching (pathway a; Fig. 6). It is also conceivable that after the initial rapid interaction, a second substrate molecule may bind to the heme while the peripheral site is still occupied (pathway b in Fig. 6). In both pathways, the last step (Fig. 6, 3) is attributed to a conformational change in the active site of the enzyme associated with a further adjustment of the iron spin equilibrium and does not result in any further quenching of the ligand fluorescence.

In order to be able to distinguish between these pathways, it is critical to determine the binding stoichiometry of substrates to P450 3A4 after equilibration of the system. The direct determination of the number of bound P450 substrate molecules is technically difficult for a number of reasons, but bromocriptine is reasonably water-soluble and can be readily quantified by fluorescence methods. We were unable to precisely determine the Kd for bromocriptine binding in the equilibrium dialysis and ITC experiments (Fig. 4), although the effective Kd is clearly in the low micromolar range. The unimolecular binding stoichiometry observed in these experiments is consistent with the pathway a (Fig. 6) but not with pathway b. It is important to note, however, that under these conditions, only the higher affinity site (the heme binding site) would be occupied, and at higher ligand concentrations both the peripheral and heme binding site may be occupied spontaneously.


Figure 7
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  		FIGURE 7.
DynaFit modeling of bromocriptine binding kinetic data. A, fit to a three-step model. {Delta}A390 – A420 data for bromocriptine binding at (0.25, 0.5, 0.75, 1, 1.25, 1.5 µM) were fit globally to a three-step binding model (Scheme 1A) using the simulation software DynaFit with the rate constants k1 = 4 x 106 M–1 s–1, k–1 = 4s–1, k2 = 0.22 s–1, k–2 = 0.39 s–1, k3 = 0.15 s–1, k–3 = 0.04 s–1. The extinction coefficients for the species LE and LE* were assumed to be equal. B, fit to a two-step model (Scheme 1B). {Delta}A390 – A420 data for bromocriptine binding (as in A) were fit globally to a two-step binding model (Scheme 1B), and the rate constants k1 = 6 x 104 M–1 s–1, k–1 = 0.3 s–1, k2 = 0.15 s–1, and k–2 = 0.04 s–1 were derived from the fitting. C, fit to a two-step model (Scheme 1C). {Delta}A390 – A420 data for bromocriptine binding (as in A) were fit globally to a two-step binding model (Scheme 1C) with the rate constants k1 = 4 x 106 M–1 s–1, k–1 = 4s–1, k2 = 0.12 s–1, k–2 = 0.06 s–1.

 


Figure 8
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  		SCHEME 1.
Three possible kinetic models for ligand binding to P450 3A4. The species with an absorbance {lambda}max at 390 nm are circled, indicating an absorbance change in the reactions. See "Discussion."

 
In order to characterize the feasibility of the proposed model, the binding kinetics of bromocriptine were modeled using DynaFit software. Kinetic traces ({Delta}A390 – A420) at different bromocriptine concentrations were fit globally to the proposed model, using the solutions of the differential equations for the binding steps for pathway a (Fig. 6, 2a), and satisfactory fits were obtained (Fig. 7A and Scheme 1). The rate constants for the first step were estimated from fluorescence quenching experiments at increasing bromocriptine concentrations (k1 = 4.0 x 106 M–1 s–1, k–1 = 4s–1) and held constant during the fitting process. The results from the work with {alpha}-naphthoflavone (k1 = 2.6 x 107 M–1 s–1) also indicate that the initial step should be at least this fast.

The rate constant for the initial encounter is comparable with the reported rate constants for camphor binding to P450 101A1 (4.6 x 106 M–1 s–1) (58) and coumarin binding to P450 2A6 (2.7 x 106 M–1 s–1) (53). A Kd value of 0.5 µM was calculated based on the forward and reverse rate constants obtained for all of the three steps, which is in close agreement with the Ks value (0.4 µM) estimated for bromocriptine by spectral binding titration. A two-step model (Scheme 1B), with both steps causing a change in absorbance, could produce a satisfactory fit (Fig. 7B). However, the rate constant for the first step was unexpectedly slow (k1 = 6 x 104 M–1 s–1, k–1 = 0.3 s–1) and inconsistent with the rapid quenching observed in fluorescence experiments and the previously reported binding rate constants for other P450s (53, 57, 58). When the last step was removed from the model (Scheme 1C), the fits were unsatisfactory, supporting the existence of a third step in the binding process (Fig. 7C).

Although bromocriptine is relatively large (Mr 655) and any conclusions may not be directly applicable to smaller substrates, equally good fits (albeit with different rate constants) could be obtained for testosterone, suggesting the general suitability of the model for substrate binding to P450 3A4 (Fig. 8 and supplemental data). However, without further experiments with various substrates, it is not possible to exclude other models, including the simultaneous occupancy of the active site by multiple ligands. In this regard, a multiple ligand binding model resulted in good fits for testosterone binding data (supplementary data), although the validity of model relative to the others cannot be documented. Our efforts to conduct equilibrium dialysis or ITC experiments with testosterone have failed due to the low affinity and low solubility of this substrate in aqueous medium. Also, probing of a "silent" initial encounter between P450 3A4 and testosterone was not feasible due to the lack of testosterone fluorescence.3


Figure 9
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  		FIGURE 8.
DynaFit modeling of testosterone binding kinetic data. A, fit to a three-step model. {Delta}A390 data for testosterone binding at 10, 20, 40, 60, 80, and 100 µM were fit globally to a three-step binding model (Scheme 1A) using the simulation software DynaFit with the rate constants k1 = 4 x106 M–1 s–1, k–1 = 490 s–1, k2 = 12 s–1, k–2 = 25 s–1, k3 = 1.8 s–1, k–3 = 3.2 s–1. The extinction coefficients for the species LE and LE* were assumed to be equal. B, fit to a two-step model (Scheme 1B). {Delta}A390 data for testosterone binding (as in A) were fit globally to a two-step binding model (Scheme 1B), and the rate constants k1 = 1.7 x 105 M–1 s–1, k–1 = 14 s–1, k2 = 2s–1, and k–2 = 1.9 s–1 were derived from the fitting. C, fit to a two-step model (Scheme 1C). {Delta}A390 data for testosterone binding (as in A) were fit globally to a two-step binding model (Scheme 1C) with the rate constants k1 = 4 x 106 M–1 s–1, k–1 = 4 s–1, k2 = 7 s–1, k–2 = 10 s–1.

 
For all of the substrates studied, we attribute the last step of binding to a conformational change in the enzyme active site, possibly resulting in an increase of A390 and a decrease of A418, which would be expected from a substrate molecule displacing H2O as the sixth ligand to the heme iron (55). However, in the case of flavone, a slow decrease in A390 accompanied by an increase in A418 was observed following the faster phase, which showed the opposite pattern (Fig. 2). Considering the cooperative binding characteristic of flavone, this unexpected "reverse Type I" change (presumably conformational) may have significance in explaining the heterotropic cooperativity exerted by some ligands. If, indeed, the model proposed for bromocriptine (Fig. 6) has general applicability, the "reverse Type I" change observed for flavone will correspond to a conformational change (3 in Fig. 6) that differs structurally from the conformational change observed in the presence of bromocriptine.

One alternate explanation for our multiphasic results that can be proposed is preexisting populations of P450 3A4 that are not in rapid equilibrium and that interact with a ligand at different rates, yielding the apparent biphasic heme absorbance kinetics (e.g. Fig. 1). These parallel reactions are effectively what are proposed in the work of Friedman (66–68) and Davydov and Halpert (69), in the latter case being due to the presence of multiple oligomeric species. In principle, one could presumably fit the data of Fig. 7 to a set of multiple populations with individual rate constants and only a single reaction step, if not with two populations then with more. However, such a model does not explain the faster rates seen with the fluorescence measurements, which clearly indicate that a faster step must precede the slower steps detected by absorbance changes. The increase in fluorescence that occurs upon mixing a bound P450 3A4-bromocriptine (or {alpha}-naphthoflavone) complex with a nonfluorescent ligand (indinavir or testosterone) provides evidence that the process is reversible and argues against the fluorescence decrease being an artifact. Another point arguing in favor of a sequential process is the nature of the results seen with flavone (Fig. 2), where the spectral changes "reverse" with time. Such a pattern could not be explained by subpopulations of P450 3A4 undergoing the same reaction.

The reduction of P450 3A4 is generally quite slow in the absence of substrate (70) (except in some rather artificial systems in which a very large excess of NADPH-P450 reductase is overexpressed (63)). As pointed out already, one issue is where the substrate is when the reduction is stimulated (Fig. 6) and what changes in P450 3A4 are critical for rapid reduction. The distinction between the rates for the individual substrate binding steps allowed for an analysis. The rate of reduction measured in an experiment with bromocriptine premixed with P450 3A4 was 3.9 s–1 (Fig. 5A); the rate measured with bromocriptine in the syringe with the NADPH, not with the P450 3A4, was 0.2 s–1 (single exponential fit is not shown). Thus, we conclude that the last step of binding has to be completed (3 in Fig. 6) for the stimulation of P450 3A4 reduction, at least in the case of bromocriptine.

In light of the many published papers on the basis of cooperativity of P450 3A4 and other P450s, one question is how the work presented here differs from previous studies and models. Other articles have dealt with models involving either multiple ligands bound to P450 3A4 (34, 37–39, 41), multiple conformational states in equilibrium (66–68), or a combination of both (71). A variant on the second theme is a recent proposal involving equilibrium among multiple oligomeric states (69), although exactly how such a phenomenon relates to ligand cooperativity is unclear.4 Most of the models presented to date have utilized rapid equilibrium assumptions. What is different here, from almost all of the other models, is the emphasis on the kinetics and dynamics of substrate binding, a multistep process that is slow enough to lead to perturbation of subsequent kinetic events (e.g. Fig. 5).

The structural basis for the phenomena under investigation here is not established. Some amino acid residues contributing to cooperativity have been identified using site-directed mutagenesis (72–74). Even when cooperativity has been altered in site-directed mutagenesis experiments, there is difficulty in establishing quantitative relationships, and the free energy changes associated with small functional changes (e.g. 2–3-fold) are difficult to assign to specific roles for individual amino acids. One issue is that, if our model shown in Fig. 6 has validity, many residues are involved in the entire pathway of substrate binding and steering to the active center. Another issue is that residues removed from the active site may be important in conformational changes that may be necessary (e.g. in hinge regions).

Another consequence of the model shown in Fig. 6 is that individual substrates may come to equilibrium with varying populations in different parts of the protein. The possible relevance of the P450 crystal structure with progesterone docked at a peripheral part of the protein (11) may be one example. Another is ethylmorphine, one of the P450 3A4 substrates for which cytochrome b5 does not show rate enhancement (70). Ethylmorphine does not produce a Type I binding spectrum or change the oxidation-reduction potential but still stimulates the rate of reduction of P450 3A4 (rate of 22 s–1) (48, 63, 70). These phenomena could be explained by a model (Fig. 6) in which the residency of the ligand is skewed toward occupancy away from the heme iron.

In conclusion, we have observed that the kinetics of binding of ligands to P450 3A4 are slower and more complex than can be explained by simple two-state systems that apply to other mammalian P450s (51, 53, 57, 58). The results obtained for bromocriptine are best explained by a process that involves three or possibly more steps (Fig. 6 and Scheme 1). We propose that these events, the slower of which have rate constants on the same time scale as subsequent steps in catalysis, are an integral part of the complexity and cooperative behavior of P450 3A4. In this regard, the finding that the apparent n value for the cooperativity of testosterone 6beta-hydroxylation was reproducibly lowered by deuterium substitution (19) may be a reflection of this scrambling of rates of substrate interactions with reduction and subsequent catalytic steps.


   FOOTNOTES
 
* This work was supported in part by United States Public Health Service Grants R01 CA090426 and P30 ES000267. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains a plot of binding of flavone to P450 3A4, second-order plots for binding of the ligands presented in Table 1 to P450 3A4, midazolam binding kinetic data fit to the models shown in Scheme 1, testosterone binding kinetic data fit to a three-step model with two ligands, and sample DynfaFit scripts for bromocriptine binding kinetic data fit to a three-step model. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University School of Medicine, 638 Robinson Research Bldg., 23rd and Pierce Aves., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: f.guengerich{at}vanderbilt.edu.

2 The abbreviations used are: P450, cytochrome P450 (also termed "heme thiolate P450" (1)); ITC, isothermal titration calorimetry. Back

3 Recently, Roberts et al. (44) used EPR binding studies with high concentrations of P450 3A4 to suggest that the first bound molecule of testosterone does not result in a spin shift and, hence, cannot be detected by absorbance. A second molecule of testosterone binds to P450 3A4 with a lower affinity than the first one, which can be characterized as negative cooperativity in this model (44). Another study, using monomeric P450 3A4 in artificial membrane "Nanodiscs®," also provided evidence that the first testosterone molecule does not produce a heme perturbation (43). A potential concern about the interpretation of the results of Roberts et al. (44), in which EPR studies were done at 77 K and compared with optical data at 298 K, is the possibility of temperature-dependent spin equilibria, for which evidence has been presented for other mammalian P450s (65). Roberts et al. (44) did not find differences in the P450 3A4 spin states in comparing 77 versus 230 K (EPR) or 1 versus 25 °C (absorbance). Back

4 In the recent report mentioned (69), the presence of the substrate bromocriptine attenuated the rate of reduction of P450 3A4 by the artificial chemical reductant Na2S2O4, but in our own work, the rate of P450 3A4 reduction by the physiological reductant NADPH-P450 reductase was accelerated by bromocriptine, as expected. The reduction of P450 3A4 by the reductase has not, to our knowledge, been studied in the Nanodisc® monomeric system. Back


   ACKNOWLEDGMENTS
 
We thank L. Mizoue (Center for Structural Biology, Vanderbilt University) for assistance with the ITC instrument, M. V. Martin and W. A. McCormick for expression and purification of P450 3A4, G. A. Marsch for helpful discussions regarding fluorescence experiments, and K. Trisler for assistance in preparation of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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