(Received for publication, October 17, 1994; and in revised form, December 15, 1994)
From the
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 17-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.
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.
where A
is the total absorbance change
observed at time t, a
is the absorbance
change for kinetic component i at t = 0, k
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 A
` and
A
are the absorbance changes observed at time t for the
reactions in the presence and absence of substrate. a
` and a
are the absorbance
changes, and k
` and k
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.
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 17ethynylestradiol 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)
17
-ethynylestradiol (0.036 s)
cimetidine (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 , k
, and k
relative to those obtained with no addition. However, the values
of the absorbance parameters a
and a
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 and k
`, 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
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
and k
(Table 2), which correspond to the substrate-specific P450
3A4 species in the absence of substrate. Results with
17
-ethynylestradiol are not presented since the data did not fit using either a one- or two-species model, and
17
-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 = 3.2 s
) binds
nifedipine while a more rapid P450 species (k
= 41.4 s
) is recognized by quinidine;
these species are also present at comparable levels (a
= 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 to k
` 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`
and a
, 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
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
=
0.0099 and 0.0051, respectively). In addition, the ratio of a
` to a
can be used to gauge
the effect of substrate on the photodissociation efficiency of a target
P450 species. Thus testosterone increased the efficiency (a
= 0.0099 and a
`
= 0.0141) while warfarin considerably decreased the efficiency (a
= 0.0060 and a
`
= 0.0033), and nifedipine, erythromycin, and quinidine had no
effect. Although, as mentioned above, the difference kinetic parameters
for 17
-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 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
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 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,
with the latter a requirement for steroid 6
-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.