©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CO Binding Kinetics of Human Cytochrome P450 3A4
SPECIFIC INTERACTION OF SUBSTRATES WITH KINETICALLY DISTINGUISHABLE CONFORMERS (*)

(Received for publication, October 17, 1994; and in revised form, December 15, 1994)

Aditya P. Koley (1) Jeroen T. M. Buters (1) Richard C. Robinson (1) Allen Markowitz (2) Fred K. Friedman (1)(§)

From the  (1)Laboratory of Molecular Carcinogenesis, NCI and (2)Biomedical Instrumentation and Engineering Program, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The kinetics of CO binding to human cytochrome P450 3A4 was examined by the flash photolysis technique, employing the membrane-bound P450 expressed in baculovirus-infected SF9 insect cells. Triexponential kinetics was observed, indicating that P450 3A4 is composed of multiple, kinetically distinguishable conformers. To define the substrate specificity of individual P450 3A4 conformers we evaluated the effect of a series of substrates of varying sizes and structures on the CO binding kinetics. The rate of CO binding to the total mixture of P450 3A4 conformers was increased in the presence of nifedipine and erythromycin, decreased by quinidine, testosterone, and warfarin, and unaffected by cimetidine and 17alpha-ethynylestradiol. A recently developed kinetic difference method (Koley, A. P., Robinson, R. C., Markowitz, A., and Friedman, F. K.(1994) Biochemistry 33, 2484-2489) was used to define the kinetic parameters of individual P450 3A4 conformers. The results showed that different conformers have distinct substrate specificities. The substrates had markedly variable effects on the CO binding kinetics of their target P450 3A4 conformers and thus differentially modulate their conformations. These results demonstrate that the interaction of a particular substrate with a specific P450 3A4 conformer can be assessed in the presence of multiple conformers.


INTRODUCTION

The cytochromes P450 catalyze the oxidation of a wide variety of xenobiotic and endogenous compounds(1, 2, 3, 4) . The different forms of P450 display unique substrate and catalytic activity profiles. In particular, the 3A subfamily metabolizes numerous substrates of varying sizes and structures (reviewed in (5) ). One of the most versatile and abundant P450s in human liver is the 3A4 form(6) , whose many structurally diverse substrates ( (7) and references therein) include the drugs nifedipine (8) and quinidine(9, 10) , the macrolide antibiotic erythromycin(9, 11) , steroids such as testosterone(8, 12, 13) , and the immunosuppressive agent cyclosporin(11, 12, 14) .

To gain insight into the basis for P450 3A4 recognition of such structurally diverse substrates we examined the effects of selected substrates (Fig. 1) of varying sizes and structures on the kinetics of CO binding to P450 3A4 expressed in baculovirus-infected SF9 insect cells, employing the flash photolysis technique. Since the kinetics reflects the rate of CO diffusion through the protein matrix to the heme iron, it is a sensitive probe of P450 conformation, which provides valuable information about the heme environment, protein structure/dynamics, and the mode of substrate binding ( (15) and references therein). Although we used a single expressed P450, the results reveal it is kinetically heterogeneous and thus is composed of multiple conformers. A recently developed kinetic difference method (15) was applied to define the CO binding kinetics of different P450 3A4 conformers. This approach revealed that various substrates differentially interact with and regulate binding of the CO ligand to different 3A4 conformers and specifically modulate protein-assisted positioning of substrate, ligand, and heme in the P450 active site.


Figure 1: Substrates evaluated for their effect on the CO binding kinetics of expressed human P450 3A4.




MATERIALS AND METHODS

Human P450 3A4

This P450 was expressed in SF9 insect cells using a recombinant baculovirus as described(16) . Cells were harvested by centrifugation, washed, suspended in phosphate-buffered saline, and stored at -80 °C until use. This stock was thawed and homogenized before use. The total P450 content was spectrally determined(17) , and the protein content measured with bicinchoninic acid(18) .

CO Flash Photolysis

Reactions were carried out using 0.5 nmol/ml P450 3A4 (corresponding to 1.2 mg/ml cell homogenate protein) and 20 µM CO at 23 °C in phosphate-buffered saline (pH 7.1) containing 20% glycerol (w/v). When present, substrates (obtained from Sigma) were first added (from a 20 mM stock solution in methanol) to yield a final concentration of 300 µM, as initial experiments showed that this concentration produced maximal effects on the CO binding kinetics. The mixture was then incubated for 20 min prior to addition of CO. Photodissociation of the P450-CO complex and monitoring of CO binding kinetics at 450 nm were performed as described(19) .

Data Analysis

Standard multiexponential analysis of kinetic data was performed according to ,

where DeltaA(t) is the total absorbance change observed at time t, a(i) is the absorbance change for kinetic component i at t = 0, k(i) is the observed pseudo-first order rate constant for component i, and n is the number of independent components.

To kinetically distinguish the individual substrate-specific P450 species described under ``Results and Discussion'' from the total P450 species, we applied a kinetic difference method that was developed to similarly distinguish an individual P450 form in liver microsomes that contain a multiplicity of P450s(15) . This approach evaluates the difference between the kinetic profiles obtained in the presence and absence of a P450 substrate and thus effectively cancels out the contributions from P450s that do not bind the substrate. Using this approach, kinetic parameters for individual substrate-specific P450 3A4 species were obtained by least-squares fitting of the kinetic difference curves to ,

where DeltaA(t)` and DeltaA(t) are the absorbance changes observed at time t for the reactions in the presence and absence of substrate. a(i)` and a(i) are the absorbance changes, and k(i)` and k(i) are the pseudo-first order rate constants for the substrate-specific P450 in the presence and absence of substrate, respectively. Thus, when a single P450 species is perturbed (n = 1) this equation simply reduces to a difference of two exponential terms.

Least-squares analysis was performed with RS/1 software (BBN Software Products, Cambridge, MA) on a Dell 450/ME microcomputer.


RESULTS AND DISCUSSION

Typical time course curves for CO binding to expressed P450 3A4 in the absence and presence of the representative substrates nifedipine, erythromycin, and quinidine are illustrated in Fig. 2. It is evident that both nifedipine and erythromycin accelerated CO binding, albeit to different extents, while quinidine decelerated the reaction. Testosterone and warfarin also decelerated the reaction while cimetidine and 17alphaethynylestradiol had no effect (data not shown). The overall reaction rate was thus markedly substrate-dependent and increased (as gauged by half-time) in the order testosterone (0.170 s), quinidine (0.095 s), warfarin (0.076 s), no addition (0.036 s)approx17alpha-ethynylestradiol (0.036 s)approxcimetidine (0.035 s), nifedipine (0.020 s), erythromycin (0.010 s).


Figure 2: Effect of nifedipine, erythromycin, and quinidine on kinetics of CO binding to P450 3A4. Progress curves in the absence (a) and in the presence of nifedipine (b), erythromycin (c), and quinidine (d), respectively. The CO concentration was 20 µM; substrate concentration was 300 µM. P450 3A4 concentration: 0.5 nmol/ml in phosphate-buffered saline (pH 7.1) containing 20% (w/v) glycerol; temperature, 23 °C.



To more precisely define the effect of these substrates on the CO binding kinetics, the kinetic parameters were determined using the classical multiexponential analysis represented by . First the data in the absence of substrate (Fig. 2, a) are not monoexponential as would be expected for a single, kinetically homogeneous P450 but instead were well represented with a triexponential fit, whose parameters are presented in Table 1. This result indicates that the expressed P450 3A4 is comprised of three kinetically distinguishable components. Furthermore, each component may possibly be composed of multiple subcomponents corresponding to kinetically distinct P450 3A4 species.



We obtained the best multiexponential fit parameters for CO binding in the presence of each substrate shown in Fig. 1. As was observed in the absence of substrate, the data obtained in the presence of nifedipine was triexponential (Table 1). Addition of nifedipine clearly increased the values of the rate constants k(1) , k(2), and k(3) relative to those obtained with no addition. However, the values of the absorbance parameters a(2) and a(3) in the presence of nifedipine were significantly different from the values obtained in its absence. Since these parameters correspond to the relative amounts of the two faster components, their compositions of kinetically distinguishable P450 species differ in the absence and presence of nifedipine. Data obtained with the remaining substrates yielded biexponential fits, except for cimetidine, which yielded triexponential kinetics. However, the variable absorbance values further indicate that the P450 composition of each component is variable and substrate-dependent. Standard multiexponential analysis thus cannot be used to kinetically define a substrate-specific P450 species because of the contribution of multiple P450 species to the overall reaction. We previously encountered this analytic problem in a CO flash photolysis study of rat liver microsomes, which contain a multiplicity of P450 forms, and developed a kinetic difference method (15) , which employs the perturbation of CO binding kinetics by P450 substrates to kinetically distinguish individual substrate-specific P450s. This approach essentially entails evaluation of the difference between the kinetic curves obtained in the presence and absence of a substrate according to and yields the respective kinetic parameters for the substrate-specific P450.

Fig. 3shows the resultant difference curves for the data in Fig. 2along with the least squares curve fits to . The kinetic difference procedure yielded k(1) and k(1)`, which represent the CO binding rate constants for a substrate-specific P450 3A4 species in the absence and presence of the substrate, respectively. This analysis revealed (Table 2) that nifedipine accelerated the rate of CO binding to a P450 3A4 species by 35-fold (from 3.2 to 114.8 s) while quinidine decreased the rate of a species by 34-fold (from 41.4 to 1.2 s). Furthermore, the large difference between the corresponding k(1) values (3.2 and 41.4 s) clearly shows that nifedipine and quinidine interact with different P450 3A4 species. Kinetic difference analysis of the erythromycin data did not fit assuming a single P450 species but was well represented by a fit in which erythromycin accelerated the rate of two P450 species to different extents (Table 2). Difference kinetic analyses of experiments with the remaining substrates in Fig. 1were performed, with the exception of cimetidine, which had no effect on the CO binding kinetics. The results reveal that the substrates interacted with different P450 3A4 species as evidenced by the variability in the parameters a(1) and k(1) (Table 2), which correspond to the substrate-specific P450 3A4 species in the absence of substrate. Results with 17alpha-ethynylestradiol are not presented since the data did not fit using either a one- or two-species model, and 17alpha-ethynylestradiol presumably requires a higher order fit, which could not be reproducibly obtained with the signal to noise ratio of our current instrumentation.


Figure 3: Difference kinetic analysis of the CO binding curves in Fig. 2. Difference traces are shown for nifedipine (A), erythromycin (B), and quinidine (C), as calculated from the differences between curves b and a, c and a, and d and a in Fig. 2, respectively. The solidlines represent the best fit according to .





The results from the difference kinetic analysis are thus consistent with the multiexponential analyses, which indicated that the expressed P450 3A4 is composed of multiple kinetically distinct species. However the difference kinetic approach additionally demonstrated that different species can be defined by their substrate specificities and yielded the kinetic parameters of each species. For example a relatively slowly reacting P450 species (k(1) = 3.2 s) binds nifedipine while a more rapid P450 species (k(1) = 41.4 s) is recognized by quinidine; these species are also present at comparable levels (a(1) = 0.0061 and 0.0051, respectively).

The substrates also markedly differed in their degree of acceleration or inhibition of CO binding to their target P450 species, as evidenced by comparing k(1) to k(1)` for each substrate. We interpret these findings in terms of a dual mechanism of substrate action(15) ; a substrate can 1) change the P450 conformation/dynamics of the ligand access channel and/or 2) sterically hinder diffusion of CO to the heme iron. The first mechanism either hinders or accelerates CO binding while the second mechanism can only reduce the CO binding rate. The first mechanism thus predominates for nifedipine and erythromycin, which accelerated CO binding to their respective target P450 3A4 species, while the first and/or second factor is operative for quinidine, testosterone, and warfarin, which decreased the rate of their target P450 species.

In addition to the effects of these substrates on the rate of CO binding, Table 2shows the substrate sensitivity of a(1)` and a(1), parameters that correspond to the amount of P450 3A4 that is CO-reactive in the respective presence and absence of a given substrate. The variability in a(1) thus reflects the different amount of each substrate-specific P450 3A4 species; for example, there is twice as much testosterone as quinidine-specific P450 3A4 species (a(1) = 0.0099 and 0.0051, respectively). In addition, the ratio of a(1)` to a(1) can be used to gauge the effect of substrate on the photodissociation efficiency of a target P450 species. Thus testosterone increased the efficiency (a(1) = 0.0099 and a(1)` = 0.0141) while warfarin considerably decreased the efficiency (a(1) = 0.0060 and a(1)` = 0.0033), and nifedipine, erythromycin, and quinidine had no effect. Although, as mentioned above, the difference kinetic parameters for 17alpha-ethynylestradiol-specific P450 species could not be calculated, this substrate decreased the total P450 3A4 absorbance change from 0.0241 to 0.0178 (obtained by summing the absorbance values for the multiexponential fits in Table 1) and thus decreased the photodissociation efficiency of its target P450 species.

Our finding that P450 3A4 consists of a population of kinetically distinct species may be interpreted in terms of the current view that proteins exist in a spectrum of conformational substates(20) . This has been elegantly demonstrated for myoglobin(21) , which has two distinct macrostates: ligand bound and unbound. Each macrostate is comprised of a spectrum of conformational substates with slightly different CO binding rates. P450 3A4 may likewise be represented by two macrostates, substrate bound and substrate free, with each composed of multiple substates. These substates essentially represent a set of conformations, or conformers, which are kinetically distinct and differ in their substrate binding specificities. Each of the substrate-specific P450 3A4 species identified by the kinetic difference method thus corresponds to one or more closely related conformers. The magnitude of a(1) then reflects the number of conformers sensitive to a given substrate; those substrates reacting with a broader spectrum of P450 3A4 conformers would yield a higher a(1) value. This representation of P450 3A4 by a population of distinct conformers, which differ in their substrate specificities, thus provides a basis for its recognition of a variety of structurally dissimilar substrates.

The P450 3A4 conformers may differ not only in the orientation but also in the identity of the amino acid residues that bind a given substrate. That distinct substrates can bind different residues is supported by a recent kinetic study of the metabolism of 7,8-benzoflavone and polycyclic aromatic hydrocarbons by human P450 3A4(22) , which showed that these substrates simultaneously bind to different and perhaps adjacent sites. Related work on the mechanism of 7,8-benzoflavone-mediated activation of substrate metabolism by rabbit P450 3A6 (23, 24) also indicated that this flavone binds at a site distinct from the substrate binding site. These studies also suggest the possibility that in addition to the presence of conformers, multiple binding sites on a single P450 molecule offer an additional mechanism for the recognition of diverse substrates. However, the differences in the k(1) values observed with a representative set of P450 3A4 substrates (Table 2) can only originate from different P450 conformers, as these values (which represent the CO binding rate for the substrate-free P450) would be the same if these substrates bind a single P450 molecule at different sites. We thus propose that a multiplicity of conformers is the predominant factor that confers a wide range of substrate recognition by P450 3A4.

P450 3A4 activity is dependent on lipid, NADPH-cytochrome P450 reductase, and cytochrome b(5), with the latter a requirement for steroid 6beta-hydroxylation and nifedipine N-oxidation(14) . Our results implicate the conformer distribution of a P450 macrosystem as an additional element that modulates activity. Further work is planned to identify the factors that regulate the conformer distribution of P450 3A4 and whether distinct conformers are also observed using other expressed P450 forms.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Bldg. 37, Rm. 3E-24, NIH, Bethesda, MD 20892. Tel.: 301-496-6365; Fax: 301-496-8419; fkfried{at}helix.nih.gov.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.